Perfusion bioreactor

ABSTRACT

In some embodiments the present invention provides perfusion bioreactors and cell culture scaffolds suitable for the preparation of tissue grafts, such as bone tissue grafts. In some embodiments, the perfusion bioreactors comprise a graft chamber and/or a graft chamber insert configured to hold a tissue graft having a certain shape and/or certain dimensions, and/or to allow culture of such tissue grafts under press-fit direct perfusion conditions. In some embodiments, the perfusion bioreactors comprise an equilibration chamber.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/959,950 filed Dec. 4, 2015, now pending; which claims the benefitunder 35 USC § 119(e) to U.S. Application Ser. No. 62/087,614 filed Dec.4, 2014. U.S. application Ser. No. 14/959,950 filed Dec. 4, 2015 is alsois a continuation-in-part application of International Application No.PCT/US2014/072579 filed Dec. 29, 2014, now expired; which claims thebenefit under 35 USC § 119(e) to U.S. Application Ser. No. 62/087,614filed Dec. 4, 2014 and to U.S. Application Ser. No. 61/921,915 filedDec. 30, 2013, both now expired. The disclosure of each of the priorapplications is considered part of and is incorporated by reference inthe disclosure of this application.

COPYRIGHT AND INCORPORATION BY REFERENCE

A portion of the disclosure of this patent document contains materialthat is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

BACKGROUND INFORMATION Field of the Invention

The present invention relates generally to cell culture, and moreparticularly to a bioreactor and method of use thereof.

Background of the Invention

The human skeleton consists of 206 distinct bones, which support andprotect the body, and play a role in metabolism, calcium storage andblood cell production. Despite its ability to remodel throughout ahuman's lifetime and its self-healing properties, reconstructivetherapies are needed to restore functionality in clinical conditionscharacterized by large skeletal defects resulting from congenitaldisorders, degenerative diseases and trauma (Braddock, M., Houston, P.,et al. Born again bone: tissue engineering for bone repair. News PhysiolSci 2001, 16, 208-213). The economic burden of skeletal defects ismassive and expected to rapidly increase over the next decades due tothe rapid global population growth and extension of life expectancy(Hollinger, J. O., Winn, S., et al. Options for tissue engineering toaddress challenges of the aging skeleton. Tissue Eng 2000, 6, 341-350),with a combined annual US market for bone repair and regenerationtherapies projected to reach 3.5 billion by 2017 (U.S. Markets forOrthopedic Biomaterials for Bone Repair and Regeneration. MedTechInsight 2013). A large number of bone substitute materials are currentlyavailable for skeletal reconstruction, with transplantation of bonegrafts still remaining the gold standard treatment (Albert, A.,Leemrijse, T., et al. Are bone autografts still necessary in 2006? Athree-year retrospective study of bone grafting. Acta Orthop Belg 2006,72, 734-740). Nevertheless, current treatments for patients in need ofcomplex skeletal reconstruction have never reached full clinicalpotential and can be associated with life-threatening complications. Theengineering of viable bone substitutes using a combination ofpatient-specific cells and compliant biomaterial scaffolds thereforerepresents a promising therapeutic solution.

Traditional attempts to grow bone grafts in the laboratory were based onculturing cell/scaffold constructs under static conditions in thepresence of osteogenesis-inducing factors. However, static cultures arenot optimal to grow centimeter-sized bone grafts for clinicaltranslation due to poor nutrient supply and removal of metabolic waste.Under these conditions, in fact, mass transport occurs only viadiffusion, which is not sufficient to support cell survival andproliferation inside the core of large cell/scaffold constructs,resulting in necrosis and poor tissue formation. In addition, cellproliferation and matrix synthesis at the construct periphery over theculture period further impede medium diffusion and contribute to theformation of a nutrient gradient that drive cell migration towards thesubstitute borders (Goldstein, A. S., Juarez, T. M., et al. Effect ofconvection on osteoblastic cell growth and function in biodegradablepolymer foam scaffolds. Biomaterials 2001, 22(11), 1279-1288). On top ofthis, culture in static conditions does not allow provision of thosebiophysical stimuli that are critical for functional regeneration(Yeatts, A. B., Fisher, J. P. Bone tissue engineering bioreactors:dynamic culture and the influence of shear stress. Bone 2011, 48(2),171-181; Klein-Nulend, J., Bakker, A. D., et al. Mechanosensation andtransduction in osteocytes. Bone 2013, 54(2), 182-190). Advances inbioreactor systems over the last two decades have opened newopportunities in the field of bone engineering as they allow to nurturethe development of bone tissue by providing an appropriate physiologicalenvironment with stimulatory biochemical and biophysical signals(Salter, E., Goh, B., et al. Bone tissue engineering bioreactors: a rolein the clinic? Tissue Eng Part B Rev 2012, 18(1), 62-75).

Bioreactors were initially developed to allow the high-mass culture ofcells used for applications in diverse areas, including fermentation,wastewater treatment and purification, food processing and drugproduction (Martin, I., Wendt, D., et al. The role of bioreactors intissue engineering. Trends Biotechnol 2004, 22(2), 80-86). Many of theprinciples established by these applications have recently been adaptedfor tissue engineering purposes. A bioreactor for tissue engineeringapplications should (i) facilitate uniform cell distribution, (ii)provide and maintain the physiological requirements of the cell (e.g.,nutrients, oxygen, growth factors), (iii) increase mass transport bothby diffusion and convection using mixing systems of culture medium, (iv)expose cells to physical stimuli, and (v) enable reproducibility,control, monitoring and automation. The ultimate design of a tissueengineering bioreactor is application specific, but basiccharacteristics are required when engineering tissue substitutes forpotential clinical applications, such as the use of materials that donot release toxic products and can withstand numerous cycle of hightemperature and pressure for repeated steam sterilization in autoclaves.Furthermore, bioreactors should present a simple design in order toprevent contamination and allow quick access to the engineered tissue ifany problem arises in the system during the operational period (e.g.,fluid leakage and flow obstruction). Despite the fact that severaldesign solutions and range of stress values imparted to the cells havebeen explored to date, bioreactors for bone engineering applications arebroadly classified in few main categories, including rotating wallvessels, spinner flasks, perfusion bioreactors and compression systems(for review, see Sladkova and de Peppo (2014) Bioreactor systems forhuman bone tissue engineering, Processes 2(2) 494-525.).

Perfusion bioreactors for bone engineering applications are culturesystems composed of several key elements, including one or more chamberswhere the cell/scaffold constructs are placed, a medium reservoir, atubing circuit and a pump enabling mass transport of nutrients andoxygen throughout the perfusion chamber. Perfusion bioreactors arebroadly classified into indirect or direct systems, depending on whetherthe culture medium is perfused around or throughout the cell/scaffoldconstructs.

In indirect perfusion bioreactors, the cell/scaffold constructs areloosely placed in the equilibration chamber, and the culture mediumpreferentially follows the path of least resistance around theconstructs, resulting in reduced mass transfer throughout the core ofthe samples. Therefore, the convective forces generated by the perfusionpump mitigate the nutrient concentration gradients principally at thesurface of the cell/scaffold constructs, thus limiting the size of bonesubstitutes that can be engineered using these systems. On the otherhand, indirect perfusion bioreactors may represent valuable systems forthe collective culture of a large number of small particulatecell/scaffold constructs that can be then assembled to repair large andgeometrically complex skeletal defects (de Peppo, G. M., Sladkova, M.,et al. Human embryonic stem cell-derived mesodermal progenitors displaysubstantially increased tissue formation compared to human mesenchymalstem cells under dynamic culture conditions in a packed bed/columnbioreactor. Tissue Eng Part A 2013, 19, 175-187; David, B.,Bonnefont-Rousselot, D., et al. A Perfusion Bioreactor for EngineeringBone Constructs: An in Vitro and in Vivo Study. Tissue Eng Part CMethods 2011, 17(5):505-516).

In direct perfusion bioreactors, the cell/scaffold constructs are placedin the equilibration chamber in a press-fit fashion so that the culturemedium is forced to pass through the center of the samples. In view ofthis advantage, direct perfusion bioreactors have been used to engineerbone substitutes using a combination of different human osteocompetentcells and biomaterial scaffolds (for review, see Sladkova and de Peppo(2014) Bioreactor systems for human bone tissue engineering, Processes2(2) 494-525.). Studies demonstrate that direct perfusion of differentcombinations of cell/scaffold constructs highly support cell survivaland proliferation, and formation of mature bone-like tissue, thusrepresenting an optimal culture system for the construction of relevantbone substitutes with potential in clinical application of skeletalreconstructions.

While biomimetic tissue engineering strategies have been explored for exvivo cultivation of functional bone substitutes by interfacingosteocompetent cells to biomaterials under appropriate cultureconditions in bioreactors, engineering large and geometrically complexbone grafts for extensive skeletal reconstructions remains problematicusing current engineering approaches. In particular, as discussed above,culture of large bone grafts is problematic using common perfusionbioreactors, due to the flow resistance caused by the large size of thegraft. The development of newly formed bone tissue progressively limitsthe medium perfusion, with negative consequences on the functionality ofthe perfusion system and graft viability. Thus there remains a need fornew approaches and tools to facilitate the in vitro preparation offunctional bone tissue and large bone grafts. Such new approaches andtools could also be used for the in vitro preparation of other types oftissue grafts, other than bone.

SUMMARY OF THE INVENTION

Some of the main aspects of the present invention are summarized below.Additional aspects of the present invention are described in theDetailed Description of the Invention, Examples, Drawings and Claimssections of this patent application. The description in each of thesections of this patent application is intended to be read inconjunction with the other sections. Furthermore, the variousembodiments described in each of the sections of this patent applicationcan be combined in various different ways, and all such combinations areintended to fall within the scope of the present invention.

To overcome the obstacles of current methods, the present inventionprovides perfusion bioreactors and cell culture scaffolds for growingfunctional vascularized tissues, such as bone, in vitro. The size andshape of the scaffolds and bioreactors can be customized usinginnovative engineering strategies based on a combination of medicalimaging, computer-assisted design (CAD) and/or computer-assistedmanufacturing (CAM). In addition, digital drawing and simulationsoftware can be used to optimize the design of the perfusionbioreactors, and for driving the controlled manufacturing of theperfusion bioreactors. For example, in some embodiments the presentinvention provides perfusion bioreactors and cell culture scaffolds tofacilitate segmental additive bone engineering (SABE) and/or segmentaladditive tissue engineering (SATE), which enable segments of functionalvascularized tissues, such as bone, to be grown in vitro.

In one embodiment, the invention provides a perfusion bioreactorsuitable for use in the preparation of a tissue graft segment, such as abone graft segment, comprising an equilibration chamber.

In one embodiment, the invention provides a perfusion bioreactorsuitable for use in the preparation of a tissue graft segment,comprising at least one graft chamber configured to accommodate a tissuegraft segment.

In one embodiment, the invention provides a perfusion bioreactorsuitable for use in the preparation of a tissue graft segment,comprising (i) at least one graft chamber and (ii) at least one graftchamber insert configured to accommodate a tissue graft segment.

In one embodiment, the invention provides a perfusion bioreactorsuitable for use in the preparation of a tissue graft segment, such as abone graft segment, comprising: (i) at least one graft chamberconfigured to accommodate a tissue graft segment; and (ii) at least oneequilibration chamber.

In one embodiment, the invention provides a perfusion bioreactorsuitable for use in the preparation of a tissue graft segment,comprising (i) at least one graft chamber, (ii) at least one graftchamber insert configured to accommodate a tissue graft segment, and(iii) at least one equilibration chamber.

In one embodiment, the invention provides a perfusion bioreactorsuitable for use in preparation of a tissue graft segment, comprising(i) a graft chamber; and (ii) an equilibration chamber in fluidcommunication with the graft chamber. In one embodiment, the bioreactorfurther includes an inlet, a fluid channel defining a fluid path betweenthe inlet and the equilibration chamber, a fluid reservoir, and anaperture fluidly connecting the fluid reservoir and the graft chamber,the fluid reservoir further comprising an outlet port.

In one embodiment, the invention provides a perfusion bioreactorsuitable for use in the preparation of a tissue graft segment, such as abone graft segment, comprising: (a) a bottom portion, comprising: (i) atleast one graft chamber configured to accommodate a tissue graftsegment; (ii) at least one equilibration chamber; (iii) an inlet port;(iv) a fluid channel connecting the equilibration chamber to the inletport; and (b) a top portion, comprising: (a) a fluid reservoir; (b) atleast one opening connecting the fluid reservoir and the graft chamber;and (c) an outlet port. In some such embodiments the top portion and thebottom portion can be secured together using any suitable fasteningmechanism.

In some embodiments the perfusion bioreactors described herein may beconnected to, or provided together with, a pump, and optionally also oneor more tubes to connect the pump to the bioreactor. For example, in oneembodiment, the bioreactor may be used in conjunction with, or providedtogether with, a pump, and one or more tubes connecting the inlet portand/or the outlet port to the pump.

In one embodiment, the invention provides a perfusion bioreactorsuitable for use in the preparation of a tissue graft segment, such as abone graft segment, comprising: (a) a bottom portion, comprising: (i) atleast one graft chamber configured to accommodate a tissue graftsegment, such as bone graft segment; (ii) at least one equilibrationchamber; (iii) an inlet port; (iv) a fluid channel connecting theequilibration chamber to the inlet port; and (b) a top portion,comprising: (a) a fluid reservoir; (b) at least one opening connectingthe fluid reservoir and the graft chamber; and (c) an outlet port,wherein the top portion and the bottom portion are secured together by afastening mechanism; and (c) a pump; and (d) one or more tubesconnecting the inlet port, the outlet port and the pump.

In some embodiments, the graft chamber dimensions are designed toaccommodate a particular tissue segment, such as a bone segment, forexample by using a digital three-dimensional model of the tissue/bonegraft segment to custom-design the graft chamber. The graft chamber mayhave the same size and shape as the tissue/bone segment, orapproximately the same size and shape as the tissue/bone segment, orhave a size and shape such that the tissue/bone segment will fit intothe graft chamber in a press-fit configuration. In some embodiments, thegraft chamber further comprises a frame or insert to provide and/ormaintain the desired dimensions of the graft chamber (e.g., the desiredinternal dimensions of the graft chamber, e.g., to accommodate thetissue/bone graft in a press-fit configuration) and/or maintain fluidflow through the perfusion bioreactor. In some embodiments, the frame orinsert may be made of or comprise any suitable material. For example, insome embodiments the frame or insert can be made from any material thatcan easily be molded or cut to have the desired dimensions, such as thedimensions of the tissue/bone graft. In some such embodiments thatmaterial may also be compliant, in order to allow the best fit betweenthe graft chamber and the tissue/bone graft. For example, in someembodiments the frame/insert may comprise a biocompatible, non-toxic,moldable plastic, such as silicone or a silicone-like material. In someembodiments, the frame/insert may comprise polydimethylsiloxane (PDMS),e.g., a PDMS ring.

In some embodiments, the tissue/bone graft segment has a maximumthickness of about one centimeter or less. In some embodiments, thetissue/bone graft segment has a maximum thickness of about 0.3millimeters to about 10 millimeters. In some embodiments, the digitalthree-dimensional model of the tissue/bone graft segment is generated bymedical imaging, computed tomography, computer-assisted design, or anycombination thereof.

In some embodiments, the equilibration chamber further comprises a flatfloor or a tapered floor. In some embodiments, the equilibration chamberfurther comprises diffusion frits. In some embodiments, theequilibration chamber further comprises a frame to maintain thedimensions of the equilibration chamber and/or maintain fluid flowthrough the perfusion bioreactor.

In some embodiments, the fastening mechanism comprises screws, rods,pins, clips, latches or any combination thereof. In some embodiments,the top portion and the bottom portion further comprise one or moreholes to facilitate the fastening mechanism. In some embodiments, thebottom portion further comprises a sealing device capable of preventingfluid leakage, for example, one or more o-rings or gaskets. In someembodiments, the bioreactor further comprises a gasket situated betweenthe top portion and bottom portion. In some embodiments, the pump is aperistaltic pump. In some embodiments, the top portion, the bottomportion or both are generated using computer-assisted manufacturing. Insome embodiments, the computer-assisted manufacturing comprisesthree-dimensional printing. In some embodiments the computer-assistedmanufacturing comprises a computer-numerical-control milling machine.

In one embodiment, the invention provides a perfusion bioreactorsuitable for use in the preparation of a tissue graft segment, such as abone graft segment, comprising: (a) a bottom portion, comprising: (i) atleast one graft chamber configured to accommodate a tissue graftsegment, such as a bone graft segment; (ii) at least one equilibrationchamber; (iii) an inlet port; (iv) a fluid channel connecting theequilibration chamber to the inlet port; and (b) a top portion,comprising: (a) a fluid reservoir; (b) at least one opening connectingthe fluid reservoir and the graft chamber; and (c) an outlet port,wherein the top portion and the bottom portion are secured together by afastening mechanism; and (c) a pump; and (d) one or more tubesconnecting the inlet port, the outlet port and the pump. In someembodiments, the graft chamber dimensions are the same as or similar toa digital three-dimensional model of the tissue/bone graft segment. Insome embodiments, the tissue/bone graft segment has a maximum thicknessof about one centimeter or less. In some embodiments, the tissue/bonegraft segment has a maximum thickness of about 0.3 millimeters to about10 millimeters. In some embodiments, the digital three-dimensional modelof the tissue/bone graft segment is generated by medical imaging,computed tomography, computer-assisted design, or any combinationthereof. In some embodiments, the equilibration chamber furthercomprises a flat floor or a tapered floor. In some embodiments, theequilibration chamber further comprises diffusion frits. In someembodiments, the equilibration chamber further comprises a frame tomaintain the dimensions of the equilibration chamber and/or maintainfluid flow through the perfusion bioreactor. In some embodiments, thegraft chamber further comprises a frame to maintain the dimensions ofthe graft chamber and/or maintain fluid flow through the perfusionbioreactor. In some embodiments, a frame may comprise a PDMS ring. Insome embodiments, the fastening mechanism comprises screws, rods, pins,clips, latches or any combination thereof. In some embodiments, the topportion and the bottom portion further comprise one or more holes tofacilitate the fastening mechanism. In some embodiments, the bottomportion further comprises a sealing device capable of preventing fluidleakage, for example, one or more o-rings or gaskets. In someembodiments, the bioreactor further comprises a gasket situated betweenthe top portion and bottom portion. In some embodiments, the pump is aperistaltic pump. In some embodiments, the top portion, the bottomportion or both are generated using computer-assisted manufacturing. Insome embodiments, the computer-assisted manufacturing comprisesthree-dimensional printing. In some embodiments the computer-assistedmanufacturing comprises a computer-numerical-control milling machine.

In one embodiment, the invention provides a cell culture scaffoldsuitable for use in the preparation of a tissue/bone graft segment,wherein the cell culture scaffold dimensions are the same as or similarto a digital three-dimensional model of the tissue/bone graft segment.In some embodiments, the digital three-dimensional model of the segmentof tissue/bone is generated by medical imaging, computed tomography,computer-assisted design, or any combination thereof. In someembodiments, the cell culture scaffold is generated usingcomputer-assisted manufacturing. In some embodiments, thecomputer-assisted manufacturing comprises three-dimensional printing. Insome embodiments, the computer-assisted manufacturing comprises acomputer-numerical-control milling machine. In some embodiments, thecomputer-assisted manufacturing comprises a casting technology. In someembodiments, the manufacturing comprises laser cutting. In someembodiments the manufacturing comprises computer-numerical-control lasercutting. In some embodiments, the cell culture scaffold comprises orconsists essentially of decellularized bone tissue, a natural orsynthetic ceramic/polymer composite material, a material capable ofbeing absorbed by cells, a biocompatible non-resorbable material, or anycombination thereof.

In some embodiments the methods provided by the present inventionutilize three-dimensional models of a particular tissue portion (e.g., aportion of tissue to be constructed, replaced, or repaired), in order tomake customized tissue culture scaffolds, customized tissue grafts,and/or customized bioreactors for producing such tissue grafts. In somesuch embodiments the tissue culture scaffolds, tissue grafts, and/orbioreactors are designed and produced such that they have a size andshape corresponding to that of the desired tissue portion, or a segmentthereof. In some embodiments the methods of the present inventioninvolve making tissue grafts by producing two or more tissue graftsegments that can then be assembled/connected to produce the finaltissue graft. Such methods may be referred to herein as segmentaladditive tissue engineering (SATE) methods. In addition to the variousdifferent methods provided herein, the present invention also providescertain compositions and devices, including customized tissue grafts,customized tissue culture scaffolds, customized bioreactors, customizedbioreactor graft chambers, and customized bioreactor graft chamberinserts. These and other aspects of the present invention are describedin more detail below and throughout the present patent specification.

In some embodiments, the present invention provides a method ofpreparing a tissue graft, comprising: (a) obtaining a three-dimensionalmodel of a tissue portion to be produced, replaced, or repaired, (b)partitioning the three-dimensional model into two or more modelsegments, (c) preparing two or more tissue graft segments, wherein eachtissue graft segment has a size and shape corresponding to one of themodel segments of step (b), and (d) assembling the two or more tissuegraft segments to form a tissue graft.

In embodiments, preparing a tissue graft segment comprises: (i)obtaining a scaffold, wherein the scaffold has a size and shapecorresponding to a segment of a tissue portion to be produced, replaced,or repaired (a tissue segment) or a three dimensional model thereof (amodel segment), (ii) applying one or more populations of cells to thescaffold, and (iii) culturing the cells on the scaffold using aperfusion bioreactor of the present invention to form a tissue graftsegment.

In some embodiments the present invention provides a method of preparinga tissue graft segment (for example for use in conjunction with one ofthe methods described above or elsewhere herein), wherein the methodcomprises: (i) obtaining a scaffold, wherein the scaffold has a size andshape corresponding to a segment of a tissue portion to be produced,replaced, or repaired (a tissue segment) or a three dimensional modelthereof (a model segment), (ii) applying one or more populations ofcells to the scaffold, (iii) obtaining a perfusion bioreactor comprisinga graft chamber configured to accommodate the scaffold, (for examplehaving a graft chamber or graft chamber insert having an internal sizeand shape corresponding to the scaffold), (iv) inserting the scaffoldinto the graft chamber of the culture vessel, and (v) culturing thecells on the scaffold within the bioreactor to form a tissue graftsegment.

In some embodiments the present invention provides a method of preparinga tissue graft segment (for example for use in conjunction with one ofthe methods described above or elsewhere herein), wherein the methodcomprises: (i) obtaining a scaffold, wherein the scaffold has a size andshape corresponding to a segment of a tissue portion to be produced,replaced, or repaired (a tissue segment) or a three dimensional modelthereof (a model segment), (ii) obtaining a bioreactor comprising agraft chamber configured to accommodate the scaffold, (for examplehaving a graft chamber or graft chamber insert having an internal sizeand shape corresponding to the scaffold), (iii) inserting the scaffoldinto the graft chamber of the culture vessel, (iv) applying one or morepopulations of cells to the scaffold in the graft chamber, and (v)culturing the cells on the scaffold within the bioreactor to form atissue graft segment.

In some embodiments, the present invention provides various methods ofpreparing bioreactors, bioreactor graft chambers, or bioreactor graftchamber inserts, suitable for use in preparing the tissue grafts and/ortissue graft segments described herein.

In one such embodiment, the present invention provides a method ofpreparing a bioreactor, bioreactor graft chamber, or bioreactor graftchamber insert, comprising: obtaining a three-dimensional model of atissue portion to be produced, replaced, or repaired.

In another such embodiment, the present invention provides a method ofpreparing a bioreactor of the present invention, comprising: (a)obtaining a three-dimensional model of a tissue portion to be produced,replaced, or repaired, and (b) partitioning the three-dimensional modelinto two or more segments (model segments).

In another such embodiment, the present invention provides a method ofpreparing a bioreactor of the present invention, comprising: obtaining athree-dimensional model of a tissue portion to be produced, replaced, orrepaired wherein the model has been partitioned into two or moresegments (model segments).

In another such embodiment, the present invention provides a method ofpreparing a bioreactor of the present invention, comprising: (a)obtaining a three-dimensional model of a tissue portion to be produced,replaced, or repaired, (b) partitioning the three-dimensional model intotwo or more model segments, (c) preparing two or more bioreactors,wherein each has an internal size and shape that corresponds to the sizeand shape of one of the model segments of (b).

In addition to the methods described above, numerous variations on suchembodiments are envisioned and are within the scope of the presentinvention, including, but not limited to embodiments that combine anyone or more of the methods or method steps described above, or alter theorder of any of the method steps described above.

In some embodiments, the present invention provides tissue grafts, andsegments thereof (tissue graft segments). For example, in someembodiments, the present invention provides tissue grafts and tissuegraft segments made using any of the methods described herein.

In one embodiment the present invention provides a tissue graftcomprising two or more tissue graft segments. In one embodiment thepresent invention provides a tissue graft comprising two or more tissuegraft segments, wherein the tissue graft has a shape and sizecorresponding to a tissue portion to be replaced or repaired, or athree-dimensional model thereof.

In one embodiment the present invention provides a tissue graftcomprising two or more tissue graft segments, wherein each tissue graftsegment has a maximum thickness (i.e., at its thickest point) of fromabout 0.3 millimeters to about 10 millimeters.

In one embodiment the present invention provides a tissue graftcomprising two or more tissue graft segments, wherein each tissue graftsegment comprises tissue cells differentiated from stem cells orprogenitor cells (e.g., induced pluripotent stem cells).

In one embodiment the present invention provides a tissue graftcomprising two or more tissue graft segments, wherein each tissue graftsegment comprises endothelial cells, such as endothelial cellsdifferentiated from stem cells or progenitor cells (e.g., inducedpluripotent stem cells).

In one embodiment the present invention provides a vascularized tissuegraft comprising two or more tissue graft segments, wherein each tissuegraft segment has a maximum thickness (i.e., at its thickest point) offrom about 0.3 millimeters to about 10 millimeters.

In one embodiment the present invention provides a vascularized bonegraft comprising two or more bone graft segments, wherein each bonegraft segment has a maximum thickness (i.e., at its thickest point) offrom about 0.3 millimeters to about 10 millimeters and wherein the bonegraft comprises bone cells derived from stem cells or progenitor cells(e.g., induced pluripotent stem cells) and endothelial cells derivedstem cells or progenitor cells (e.g., induced pluripotent stem cells).

In addition to the tissue grafts described above, numerous variations ofsuch tissue grafts are envisioned and are within the scope of thepresent invention, including, but not limited to those describedelsewhere in the present specification and those that combine any one ormore of the elements described above or elsewhere in the application.

In some embodiments, the present invention provides bioreactors of thepresent invention. For example, in some embodiments, the presentinvention provides bioreactors made using any of the methods describedherein.

In one embodiment the present invention provides bioreactors of thepresent invention, wherein the internal portion thereof has a size andshape corresponding to the tissue portion to be replaced or repaired, asegment of the tissue portion to be replaced or repaired, or athree-dimensional model of any thereof.

In one embodiment the present invention provides bioreactors of thepresent invention, wherein the internal portion thereof is designed toaccommodate a scaffold or a tissue graft segment that has a size andshape corresponding to a segment of a tissue portion to be replaced orrepaired.

In one embodiment the present invention provides bioreactors of thepresent invention, wherein the internal portion thereof is designed toaccommodate a scaffold or a tissue graft segment, wherein each tissuegraft segment has a maximum thickness (i.e., at its thickest point) offrom about 0.3 millimeters to about 10 millimeters.

In addition to the bioreactors described herein, numerous variations ofsuch bioreactors, bioreactor graft chambers, and bioreactor graftchamber inserts are envisioned and are within the scope of the presentinvention, including, but not limited to, those described elsewhere inthe present specification and those that combine any one or more of theelements described above or elsewhere in the application.

In some of the above embodiments, the tissue grafts or tissue graftsegments are bone tissue grafts or bone tissue graft segments. In someembodiments, the tissue grafts or tissue graft segments are cartilagegrafts or cartilage graft segments.

In some of the above embodiments, the tissue grafts or tissue graftsegments comprise mammalian cells, such as cells from non-humanprimates, sheep, or rodents (such as rats or mice). In some of the aboveembodiments, the tissue grafts or tissue graft segments comprise humancells. In some of the above embodiments, the tissue grafts or tissuegraft segments comprise one or more populations of cells derived fromthe same subject into which the tissue graft is to be implanted (i.e.,autologous cells). In some of the above embodiments, the tissue graftsor tissue graft segments comprise one or more populations of cellsderived from stem cells or progenitor cells, such as induced pluripotentstem cells.

In some of the above embodiments, the tissue grafts or tissue graftsegments are vascularized. In some of the above embodiments, the tissuegrafts or tissue graft segments comprise endothelial cells, such asendothelial cells derived from stem cells or progenitor cells, such asinduced pluripotent stem cells.

In some of the above embodiments the three-dimensional models and/ormodel segments are digital models, such as digital models that provide arepresentation of the three-dimensional structure of a tissue portion ora segment thereof.

In some of the above embodiments the tissue graft segments have athickness of about 20 millimeters or less, or 15 millimeters or less, or10 millimeters or less, for example at their thickest point. Forexample, in some of the above embodiments the tissue graft segments havea thickness of from about 0.3 millimeters to about 10 millimeters, forexample at their thickest point.

In some of the above embodiments the culture vessels are bioreactors,such as direct perfusion bioreactors. In some of the above embodimentsthe scaffolds or tissue graft segments are placed into bioreactors underpress-fit conditions. In some of the above embodiments tissue graftsegments are cultured in a bioreactor or the present invention underdirect perfusion and/or press-fit conditions.

In some of the above embodiments the scaffolds are generated orcustomized using computer assisted manufacturing, three-dimensionalprinting, casting, milling, laser cutting, rapid prototyping, or anycombination thereof.

In some of the above embodiments the bioreactors are generated orcustomized using computer assisted manufacturing, three-dimensionalprinting, casting, milling, laser cutting, rapid prototyping, or anycombination thereof.

In some of the above embodiments, the tissue grafts comprise two or moretissue graft segments connected using a biocompatible adhesive,stitches, sutures, staples, plates, pins, screws, or any combinationthereof.

In some embodiments the methods, compositions, and devices provided bythe present invention, and tissues prepared therefrom, can be useful fora variety of applications including for therapeutic purposes (such asrepairing pathological or traumatic tissue defects), cosmetic purposes,or in model systems for studying diseases or developing therapeutics.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B. Top side view of two embodiments of the customizedperfusion bioreactors. FIG. 1A: Customized perfusion bioreactors securedusing side latches. FIG. 1B: Customized perfusion bioreactors securedusing screws.

FIGS. 2A-2B. FIG. 2A: Top view of the customized bioreactor of FIG. 1A.FIG. 2B: Bottom view of the customized bioreactor of FIG. 1B.

FIGS. 3A-3B. Top side view of the latches (A) and screws (B) used tosecure the customized perfusion bioreactors of FIGS. 1A and 1B.

FIGS. 4A-4B. Top side view of the gaskets used to seal the junctionbetween bottom and top portions of the customized bioreactors of FIGS.1A and 1B. FIG. 4A: Gasket used to seal the perfusion bioreactor of FIG.1A secured with side latches. FIG. 4B: Gasket used to seal the perfusionbioreactor of FIG. 1B secured with screws.

FIGS. 5A-5D. Top side views of the bottom portion of customizedperfusion bioreactors showing the graft chamber 5, the equilibrationchamber 6 and perfusion channel 7 (between opening 13 and inlet port 9).FIG. 5A: Flat equilibration chamber for latch-secured perfusionbioreactors. FIG. 5B: Tapered equilibration chamber for latch-securedperfusion bioreactors. FIG. 5C: Flat equilibration chamber forscrew-secured perfusion bioreactors. FIG. 5D: Tapered equilibrationchamber for screw-secured perfusion bioreactors.

FIGS. 6A-6D. Top side views of the bottom portion of the customizedperfusion bioreactors showing a circular groove 8 surrounding the graftchamber 5 used to accommodate an O-ring preventing medium leakage. FIG.6A: Flat equilibration chamber for latch-secured perfusion bioreactors.FIG. 6B: Tapered equilibration chamber for latch-secured perfusionbioreactors. FIG. 6C: Flat equilibration chamber for screw-securedperfusion bioreactors. FIG. 6D: Tapered equilibration chamber forscrew-secured perfusion bioreactors.

FIGS. 7A-7D. Top side views of the bottom portion of the customizedperfusion bioreactors showing the inlet port 9 and perfusion channel 7.FIG. 7A: Flat equilibration chamber for latch-secured perfusionbioreactors. FIG. 7B: Tapered equilibration chamber for latch-securedperfusion bioreactors. FIG. 7C: Flat equilibration chamber forscrew-secured perfusion bioreactors. FIG. 7D: Tapered equilibrationchamber for screw-secured perfusion bioreactors.

FIGS. 8A-8B. Cross sectional views of the bottom portion of thecustomized perfusion bioreactors showing the equilibration chamber 6,the graft chamber 5 and perfusion channel 7. FIG. 8A: Flat equilibrationchamber 6. FIG. 8B: Tapered equilibration chamber 6.

FIGS. 9A-9B. Schematic cross sectional views of the graft chamber 5 andequilibration chamber 6 of the customized bioreactors showing optionalelements including diffusion frits 10, graft chamber frame 5 a, and theequilibration chamber frame 6 a used to firmly secure the graft in placeand enable direct perfusion, and modulate the dimensions of the graftand equilibration chambers. FIG. 9A: Flat equilibration chamber 6. FIG.6B: Tapered equilibration chamber 6.

FIGS. 10A-10B. Top side views of the top portion of the customizedperfusion bioreactors showing the perfusion exit 13, the fluid reservoir11 and outlet port 12. FIG. 12A: Flat equilibration chamber 6 forlatch-secured perfusion bioreactors. FIG. 6B: Flat equilibration chamber6 for screw-secured perfusion bioreactors.

FIG. 11. Side view of the customized perfusion bioreactor showing thesystem of tubes 15 and peristaltic pump 16 controlling medium perfusion.

FIGS. 12A-12B. Perspective view of exemplary cell culture scaffoldsprovided by the invention. FIG. 12A: Shows an enlarged view of a singlescaffold. The scaffold can be designed and manufactured based on adigital image of a portion of tissue, as described herein. FIG. 12B:Shows multiple scaffolds of different shapes and sizes. Multiplescaffolds can be used, for example, to prepare complementary segments ofa large bone graft, as described herein.

FIGS. 13A-13B. FIG. 13A: Perspective view of the bottom portion of anexemplary multi-chamber perfusion bioreactor for the collective cultureof more than one bone segment. FIG. 13B: Perspective view of the topportion of an exemplary multi-chamber perfusion bioreactor for thecollective culture of more than one bone segment.

FIG. 14. Top panel: (i) Digital models of skeletal defects are created,segmented (here, into three segments labeled A, B and C) and used tofabricate custom-made biomaterial scaffolds and bioreactors; (ii)Example of the top portion (A) and bottom portion (B) of a perfusionbioreactor created using CAD software. Middle panel: Osteogenic andvascular progenitors are generated from hiPSC and co-cultured ontocustom-made osteoinductive scaffolds (here, on three scaffolds labeledA, B and C) in perfusion bioreactors. Bottom panel: Engineeredvascularized bone segments (here, three segments labeled A, B and C) areassembled using biocompatible bone glues and/or reinforced using 3Dprinted titanium pins and holes.

FIGS. 15A-15B. FIG. 15A: Three-dimensional digital model of a humanfemur with a digital reconstruction of a bone defect to be repaired(dark gray). FIG. 15B: Partitioning of the digital model of the bonedefect shown in FIG. 15A into five model segments (dark gray). The modelsegments can be used to drive the manufacturing of biomaterial scaffolds(light gray) having a size and shape that corresponds to each of themodel segments.

FIG. 16. Simulation studies highlighting the need for an equilibrationchamber, and revealing the effect of its geometry on medium perfusionthroughout the tissue graft. Studies were performed in ComsolMultiphysics and were essential to guide bioreactor design. Depicted areequilibration chambers have a flat bottom and equilibration chambershaving a tapered bottom.

FIG. 17. Top side view of expanded bioreactor having a top portion andbottom portion. The bioreactor includes multiple equilibration and graftchambers.

FIG. 18. Side view of expanded bioreactor of FIG. 17 having a topportion and bottom portion. The bioreactor includes multipleequilibration and graft chambers.

FIG. 19. Top side view of bottom portion of bioreactor of FIG. 17. Thebioreactor includes multiple equilibration and graft chambers connectedby a common perfusion channel.

FIG. 20. Top side view of top portion of bioreactor of FIG. 17. Thebioreactor includes multiple apertures fluidly connecting the fluidreservoir with each graft chamber.

DETAILED DESCRIPTION OF THE INVENTION

In some embodiments the present invention provides novel perfusionbioreactors and cell scaffolds that may facilitate the engineering oflarge and/or geometrically defined tissue/bone grafts. In someembodiments the size and shape of the bioreactors and scaffolds may bebased on digital models of a tissue to be repaired, e.g., a bone defect.In some embodiments the digital model of the tissue defect may bepartitioned in sequential segments using computer-aided design (CAD)software, then the models of the tissue segments may be used as areference for the computer-aided manufacture (CAM) of custom-madeperfusion bioreactors and production of scaffolds of corresponding sizeand shape. In some embodiments bioreactors may accommodate each specificcell/scaffold construct in a press-fit fashion and allow culture underdirect perfusion conditions.

In embodiments, perfusion bioreactors provided by the invention comprisea bottom portion and a top portion. It will be appreciated that thebottom portion and top portion may be a single unitary form, i.e., asingle piece, such that leakage is prevented between the bottom and topportions. In some embodiments, the bottom portion comprises one or moregraft chamber(s) (configured to accommodate one or more bone graftsegment(s)), one or more equilibration chambers, a fluid inlet port, andone or more fluid channels connecting the equilibration chamber(s) tothe fluid inlet port. The equilibration chamber will typically bepositioned beneath the graft chamber so as to ensure homogenousperfusion and support the graft. In some embodiments, the top portioncomprises a fluid reservoir (e.g., for cell culture medium), one or moreopenings (to connect the fluid reservoir (of the top portion) and thegraft chamber(s) (of the bottom portion)), and a fluid outlet port. Insome embodiments, the top portion and the bottom portion are securedtogether by a fastening mechanism (such as, for example, latches orscrews). In some embodiments, the bioreactors further comprise a pump,for example a peristaltic pump, and one or more tubes connecting theinlet and outlet ports to facilitate fluid flow through the bioreactor.In some embodiments, the bioreactors comprise one or more gaskets oro-rings or other structures capable of sealing the bioreactor and/orpreventing fluid leakage or spillage. In some embodiments, one or morediffusion enhancing elements, such as diffusion frits, can be placedinto the equilibration chamber to improve fluid flow and homogenousgraft perfusion. In some embodiments, frames or inserts, for examplePDMS frames or inserts, can be placed or inserted into the graft chamberand/or the equilibration chamber to secure the graft in place and enableculture of the tissue segment under direct perfusion, as well as tomodulate the shape and dimensions of the graft chamber and equilibrationchamber.

The customized design and manufacture of the bioreactors, and eachelement or structure of the bioreactors is described in further detailherein.

Some of the main embodiments of the present invention are described inthe above Summary of the Invention section of this application, as wellas in the Examples, Figures and Claims. This Detailed Description of theInvention section provides additional description relating to thecompositions and methods of the present invention, and is intended to beread in conjunction with all other sections of the present patentapplication, including the Summary of the Invention, Examples, Figuresand Claims sections of the present application.

Abbreviations and Definitions

The abbreviation “CAD” refers to computer-aided design.

The abbreviation “CAM” refers to computer-aided manufacture.

The abbreviation “CNC” refers to computer-numerical-control.

As used herein, the terms “cell/scaffold” and “scaffold/cell” and“cell/scaffold construct” and “cell/scaffold complex” and “scaffold/cellconstruct” and “scaffold/cell complex” are used interchangeably to referto a scaffold to which cells have been applied.

As used herein, the terms “about” and “approximately,” when used inrelation to numerical values, mean within + or −20% of the stated value.

Additional definitions and abbreviations are provided elsewhere in thispatent specification or are well known in the art.

FIGS. 1 through 13 show exemplary bioreactors and scaffolds according tothe invention. While the designs shown in the Figures are illustrativeof the various different features of the bioreactors and scaffolds ofthe invention, the invention is not limited to the specific designsprovided in the drawings. Rather, variations and modifications of thedesigns shown in the Figures are contemplated and are within the scopeof the present invention, as described herein and as would be understoodby those of ordinary skill in the art.

FIG. 1 is a perspective view of exemplary bioreactors with illustrativefastening mechanisms securing the top portion 1 to the bottom portion 2of the bioreactor—FIG. 1A shows latches 4 as the fastening mechanism. Inthis embodiment, the latches 4 are positioned on the exterior sidesurface of the top portion 1 b and the exterior side surface of thebottom portion 2 b. FIG. 1B shows screws 3 a as the fastening mechanism.In this embodiment, the screws are inserted into holes in the topsurface of the top portion 1 a and fasten into appropriately placedholes in the top surface of the bottom element (see FIGS. 5C and 5D).Further illustrated in both FIGS. 1A and 1B is an outlet port 12 in thetop portion of the bioreactor, and an inlet port 9 in the bottomportion. The outlet port 12 is illustrated here as an opening or channelthrough the side surface 1 b of the top portion that allows fluid (e.g.,cell culture medium) to exit the fluid reservoir 11, flow through a tubeor tubes (see FIG. 11), then through a perfusion channel (see FIG. 2B)to the inlet port 9, an opening or channel through the side surface 2 bin the bottom portion of the bioreactor which allow the flow of fluidsthrough the bioreactor. The fluid reservoir 11 is a chamber in the topportion formed by the interior side surface 1 d and interior bottomsurface 1 e of the top portion. Fluid, for example, cell culture mediummay be transferred to or from the fluid reservoir using pipets or thelike.

FIG. 2 is a top view (FIG. 2A) and bottom view (FIG. 2B) of an assembledbioreactor. FIG. 2A shows the top surface 1 a of the top portion of thebioreactor and latches 4 on the side surface 1 b, a fluid reservoir 11with an opening (perfusion exit) 13, and an outlet port 12. The inletport 9 of the bottom portion is also visible in FIG. 2A. FIG. 2B showsthe bottom surface 2 c of the bottom portion of the bioreactor andlatches 4 on the side surface 2 b, an internal perfusion channel 7 isillustrated that connects to the inlet port 9. The outlet port 12 of thetop portion is also visible in FIG. 2B.

FIG. 3 shows a perspective view of exemplary arrangements illustrativefastening mechanisms, latches 4 (FIG. 3A) and screws 3 a (FIG. 3B).

FIG. 4 shows an exemplary gasket to seal the junction between the topand bottom portions of a bioreactor. The gasket 14 comprises an opening14 a to accommodate the complementary openings and chambers in the topand bottom portions of the bioreactor. FIG. 4A shows a gasket suitablefor a bioreactor secured by latches. The gasket shown in FIG. 4Bcomprises holes 14 b to accommodate a bioreactor secured by screws.

FIG. 5 shows an illustrative embodiment of the bottom portion of aperfusion bioreactor. FIGS. 5A and 5C each show an exemplaryequilibration chamber 6 having a flat floor/bottom surface. FIGS. 5B and5D each show an exemplary equilibration chamber 6 having a taperedfloor/bottom surface. In some embodiments, the floor of theequilibration chamber may have any suitable shape, including but notlimited to, straight, curved, beveled, chamfered, or any other suitableshape, as desired. The bottom portion in FIGS. 5A and 5D are suitablefor fastening by latches. The bottom portions in FIGS. 5C and 5Dcomprise holes 3 b to accommodate fastening by screws. Further elementsshown in FIGS. 5A-5D include the top surface 2 a and side surface 2 b ofthe bottom portion, a graft chamber 5, an opening (perfusion exit) 13,and an inlet port 9.

FIGS. 6A-6D show a further embodiment of the bottom portions shown inFIG. 5A-5D, respectively, where an o-ring groove 8 is illustrated on thetop surface 2 a of the bottom portion.

FIGS. 7A-7D show a transparent view of the bottom portions shown inFIGS. 5A-5D, respectively, where the perfusion channel 7 is visible onthe interior of the bottom portion. The perfusion channel facilitatesfluid flow between the inlet port 9 and the equilibration chamber 6.

FIG. 8 shows a cross-section view of an exemplary bottom portion of abioreactor having a flat equilibration chamber 6 (FIG. 8A) or a taperedequilibration chamber 6 (FIG. 8B). Further elements shown in FIGS. 8Aand 8B include the top surface 2 a of the bottom portion, a graftchamber 5, and a perfusion channel 7.

FIG. 9 shows a cross section view of exemplary graft chamber 5 andequilibration chambers 6 in the bottom portion of a bioreactor. The top,middle, and bottom panels of FIG. 9A illustrate embodiments of perfusionchambers 6 having a flat floor. The top, middle, and bottom panels ofFIG. 9B illustrate embodiments of equilibration chambers 6 having atapered floor. In the embodiments show in the top panels of FIGS. 9A and9B, diffusion frits 10 are located within the equilibration chamber.Diffusion frits can be used within the context of the invention, forexample, to redirect and/or redistribute fluid in the perfusion chamberof the bioreactor to allow for optimal perfusion of the bone segment. Inthe embodiments shown in the middle panels of FIGS. 9A and 9B, exemplarydiffusion frits 10 are present in the equilibration chamber and a graftframe 5 a is shown within the graft chamber 5. In the embodiments shownin the bottom panels of FIGS. 9A and 9B, diffusion frits 10 are presentin the equilibration chamber and an exemplary graft frame 5 a is presentin the graft chamber 5, also shown is an exemplary perfusion frame 6 awithin the equilibration chamber 6. The graft frame and/or perfusionframe are used to firmly secure the growing bone segment in place(press-fit) to enable direct fluid perfusion through the bone segment,and to modulate the dimensions of the graft chamber and equilibrationchamber as desired to accommodate the size and shape of the bonesegment. The frames may be made of any suitable material includingplastic, such as biocompatible plastic, or silicone, such aspolydimethylsiloxane (PDMS).

FIG. 10 is a perspective view of exemplary top portions of a bioreactor.FIGS. 10A and 10B illustrate the top surface 1 a, exterior side surface1 b, and interior side surface 1 d of the top portion, a fluid reservoir11 comprising an opening 13 (perfusion exit), and an outlet port 12. Thetop portion shown in FIG. 10A is suitable for fastening by latches. Thetop portion in FIG. 10B comprises holes 3 b to accommodate fastening byscrews.

FIG. 11 is a side view of an exemplary perfusion bioreactor where thetop portion 1 is fastened to the bottom portion 2 with latches 4. One ormore tubes 15 can be used to connect the outlet port 12 in the topportion to the inlet port 9 in the bottom portion. A pump 16, such as aperistaltic pump, can be used to control the flow of fluid through thebioreactor.

FIG. 12 is a perspective view of exemplary cell culture scaffoldsprovided by the invention. FIG. 12A shows an enlarged view of a singlescaffold. The scaffold can be designed and manufactured based on adigital image of a bone graft segment, as described herein. FIG. 12Bshows multiple scaffolds of different shapes and sizes. Multiplescaffolds can be used, for example, to prepare complementary segments ofa large bone graft, as described herein.

FIG. 13 is a perspective view of the top portion (FIG. 13A) and bottomportion (FIG. 13B) of an exemplary multi-chamber bioreactor provided bythe invention. FIG. 13A: The bottom portion 2 comprises multiple graftchambers 5 for the collective culture of bone segments. The graftchambers are shown in various sizes and shapes as desired to accommodatethe sizes and shapes of the bone segments. Also shown are holes 3 b tofacilitate fastening by screws. FIG. 13B: The top portion 1 comprises afluid reservoir 11, an outlet port 12, and multiple openings 13 alignedwith the graft chambers in the bottom portion so as to connect the fluidreservoir to the graft chambers in the bottom portion (FIG. 13A). Alsoshown are holes 3 b to facilitate fastening by screws.

In various embodiments, the equilibration chamber of the bioreactor ofthe present invention may include a tapered or flat surface with respectto the vertical inner side wall of the equilibration chamber. FIG. 16shows equilibration chambers having both flat (90° with respect to theinner surface of the equilibration chamber) and tapered surfaces (150°and 105° with respect to the inner surface). In various embodiments, thetaper may be any angle from 90-180° with respect to the inner side wallof the equilibration chamber, for example from about 90 to 95, 100, 105,110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175,180, 185 or 90 degrees.

Perfusion Bioreactors

The present invention provides bioreactors, suitable for use in thepreparation of tissue grafts and tissue graft segments as describedherein. The bioreactors are perfusion bioreactors, for example, directperfusion bioreactors. Perfusion bioreactors for tissue engineeringapplications are culture systems that typically comprise severalelements, including, but not limited to one or more chambers wherecell/scaffold constructs are placed (referred to herein as a “graftchamber”), a culture medium reservoir, a tubing circuit, and a pumpenabling mass transport of nutrients and oxygen. Perfusion bioreactorsmay be broadly classified into indirect or direct systems, depending onwhether the culture medium is perfused around or through thecell/scaffold constructs.

With direct perfusion bioreactors, cell/scaffold constructs are placedin a suitable graft chamber in a press-fit fashion so that the culturemedium is forced to pass through the cell/scaffold construct, ratherthan around the cell/scaffold construct. Direct perfusion bioreactorshave been used to engineer bone substitutes using a combination ofdifferent human osteocompetent cells and biomaterial scaffolds.Furthermore, in the case of bone engineering, studies demonstrate thatdirect perfusion of different combinations of cell/scaffold constructscan support cell survival and proliferation, and formation of maturebone-like tissue in vitro.

In some embodiments, the present invention provides bioreactors, such asdirect perfusion bioreactors, and methods for designing and making suchnovel bioreactors. For example, in some embodiments models, such asdigital models, of tissue portions or segments thereof, as describedabove, can be used to design and manufacture bioreactors that canaccommodate one or more cell/scaffold constructs in a press-fit fashionunder direct perfusion conditions. In some such embodiments CAD files ofa tissue segment can be used to fabricate bioreactors, or graft chambersof bioreactors, or inserts for graft chambers of bioreactors, such thatthe bioreactor graft chamber has a size and geometry that iscustom-designed to correspond to that of the tissue graft or tissuegraft segment to be produced therein, and such that the scaffold and/ortissue graft/graft segment fits snugly within the bioreactor graftchamber in a press-fit configuration.

Perfusion bioreactors provided by the present invention, or the graftchambers or graft chamber inserts thereof, can be made out of anysuitable material. Materials that are suitable for the manufacture ofbioreactors, or inserts thereof, are known in the art and any suchmaterials can be used. For example, in some embodiments bioreactors, orchambers or inserts thereof, may be made of an inert metal, such asstainless steel, or made of biocompatible plastic, or any other suitablematerial known in the art. The bioreactors may be made of material thatis opaque, translucent or transparent.

In some embodiments, a bioreactor, bioreactor graft chamber, orbioreactor graft chamber insert is generated or customized usingcomputer-assisted manufacturing. For example, in some such embodimentstissue segment files can be imported into CAM software to drive thefabrication or customization of bioreactors, bioreactor graft chambers,or bioreactor graft chamber inserts capable of accommodatinggeometrically defined scaffolds and/or tissue grafts or tissue graftsegments using any suitable method known in the art, or a combinationthereof. In some such embodiments, manufacturing or customization of thebioreactor may comprise using a rapid prototyping method, using amilling machine, using casting technologies, using laser cutting, and/orusing three-dimensional printing. In some embodiments, manufacturing orcustomization of a bioreactor, bioreactor graft chamber, or bioreactorgraft chamber insert may comprise using computer-numerical-controlmethods, such as when the manufacturing or customization processinvolves laser cutting or using a milling machine. For example, in someembodiments digital models generated using CAD software, for example, asdescribed above may be processed to generate the appropriate G-Codes todrive a computer-numerical-control (CNC) milling machine (for example,Tormach, Bridgeport) and/or to select appropriate machining tool bitsand/or program machining paths to cut the bioreactor, bioreactor graftchamber, or bioreactor graft chamber insert material into the desiredshapes (e.g., complementary to the digital models of the tissuesegments). In addition, digital drawing and simulation software can beused to optimize the design of bioreactors, bioreactor graft chambers,or bioreactor graft chamber inserts, and to drive the controlledmanufacturing or customization thereof. In some embodiments bioreactors,bioreactor graft chambers, or bioreactor graft chamber inserts, can bedesigned based on digital models of tissues or tissue segments tofacilitate culturing of cells, e.g., tissue-forming cells or other cellsas described herein or known in the art, on scaffolds in order toproduce a tissue graft or tissue graft segment having a size and shapecorresponding to the complementary digital model of the tissue or tissuesegment.

In FIGS. 1-11, 13 and 17-20, the bioreactors themselves, and certainelements such as the fluid reservoirs, perfusion equilibration and graftchambers are shown as having a circular shape. However, variations inboth the shape and size of the bioreactors and any of the elementstherein are also within the scope of the invention, and any suitablyshaped and sized bioreactors or elements can be used. For example, onewould appreciate that the equilibration and graft chambers may becircular, oval, elliptical, square, triangular, tear shaped, pearshaped, or any other desired geometric shape.

In some embodiments, the perfusion bioreactors may comprise multipleperfusion channels, equilibration chambers, graft chambers, and/or anyother elements as required or needed for the collective culture of morethan one bone segment (see FIG. 13). For example, a bioreactor accordingto the invention may be configured to accommodate the culture of one,two, three, four, five, six, seven, eight, nine, ten or more bonesegments, as desired. Typically, a bioreactor will have one outlet portand one inlet port and one or more equilibration and graft chambers.FIGS. 17-20 depict a bioreactor in one embodiment of the invention whichincludes six equilibration and graft chambers which may be utilized togenerate six bone segments simultaneously. The chambers are fluidlycoupled to a single inlet and a single outlet.

Perfusion bioreactors according to the present invention may havevarious internal structural features as needed, for example, tofacilitate manufacture or assembly of the bioreactors, and/or tomaintain alignment of openings in the top and bottom portions such thatfluid flows continuously through the bioreactor. For example, thebioreactors may have internal channels, grooves, indentations, holes,walls, bars, or pins to hold elements of the bioreactor in place insidethe bioreactor and to maintain the various openings, channels andchambers in the correct position to facilitate fluid flow through thebioreactor and perfusion of the bone segment(s). In some embodiments,the bioreactors comprise a lid or cover over the fluid reservoir. Insuch embodiments, the reservoir cover is made of a material that allowsgas exchange and oxygenation of fluid but prevents contamination offluid in the reservoir. In some embodiments, the cover is attached tothe bioreactor such that the cover can be opened and closed, for exampleby a hinge. In some embodiments, the cover is not attached to thebioreactor.

In some embodiments, the bioreactors of the invention may comprise agraft chamber that is designed or customized in order to accommodate ascaffold, tissue graft, or tissue graft segment of the desired shape andsize. In one embodiment this may be achieved by designing or customizingthe bioreactor itself such that it has a graft chamber having thedesired shape and size. In another embodiment this may be achieved usinga graft chamber insert that, when placed inside a bioreactor, produces agraft chamber that has the desired shape and size. In one embodiment, abioreactor according to the present invention comprises a graft chamberof a size sufficient to accommodate a scaffold, tissue graft, or tissuegraft segment having a thickness of about 0.3 millimeters to about 10millimeters.

In some embodiments, the scaffold and/or tissue graft segment may bepositioned in the graft chamber using a graft chamber insert, which mayalso be referred to herein as a “frame.” As described above, frames orgraft chamber inserts may be used to customize the size and shape of agraft chamber and position a scaffold and/or tissue graft segment in thegraft chamber, as desired, for example in order to allow culture thetissue graft segment under direct perfusion, press-fit conditions tomaximize the flow of fluid through the scaffold and/or tissue graftsegment, and minimize the flow of fluid around the scaffold and/ortissue graft segment. In some embodiments the graft chamber may have ageneric shape or size, but one or more frames or graft chamber insertsmay be used to customize the size and shape (e.g., the internal size andshape) of the graft chamber, as desired, to accommodate the scaffoldand/or tissue graft segment. Frames or graft chamber inserts may be madeof any suitable material. For example, in some embodiments the frameand/or graft chamber insert may comprise, consist essentially of, orconsist of, a biocompatible, non-toxic, moldable plastic, such assilicone or a silicone-like material. In some such embodiments, theframe and/or graft chamber insert may comprise polydimethylsiloxane(PDMS). Frames or graft chamber inserts may be designed and manufacturedby any suitable method, including, but not limited to, the methodsdescribed herein.

In some embodiments the graft chamber may be a custom-shaped chamber(s)that accommodates the scaffold construct(s) until maturation offunctional tissue. In one embodiment, a graft chamber is of a sizesufficient to accommodate a segment of tissue (e.g., bone) having athickness of about 0.3 millimeters to about 10 millimeters.

In some embodiments, a perfusion bioreactor provided by the inventionmay comprise sealing mechanisms and/or structures and/or configurationsthat prevent leakage or spillage of fluid from the bioreactor. Forexample, one or more gaskets or o-rings or the like (see, for example,FIGS. 4 and 6). Such elements can be made of any suitable materialincluding without limitation rubber, silicone or plastic. In someembodiments, a bioreactor provided by the invention frames in graft andequilibration chambers to ensure secure fit of scaffold/bone segments;diffusion frits or other diffusion enhancing structures/materials can beinserted into the equilibration chamber to redirect fluid flow andoptimize perfusion of bone segment. Such diffusion enhancing structuresare preferably porous, and can have any suitable configuration.

The dimensions, geometry, configuration, size and/or shape of thebioreactor and any elements therein, including, for example, the graftand equilibration chambers, the size of perfusion hole, shape of thefloor of the equilibration chamber, and thickness of the diffusion fritsmay be determined or defined using, for example, computational fluiddynamics software to simulate the hydrodynamic conditions inside aproposed configuration and experimental validation. In some embodimentsof the invention, fluid turbulence inside the bioreactor may beminimized and homogenous perfusion of the cell/scaffold constructpositioned in the graft chamber may be optimized.

Customized bioreactors of the present invention may be generated using anumber of methods. Additionally, the bioreactors may be generated from asingle unitary material, such as a block, or multiple pieces such asthose depicted in the Figures as having a top portion and bottomportion. Methods of manufacture may those conventional methods known inthe art, such as, but in no way limiting, three-dimensional printing,casting, milling, laser cutting, rapid prototyping, or any combinationthereof.

In some embodiments bioreactors, bioreactor graft chambers, and graftchamber frames or inserts, as provided by the present invention, can bedesigned and manufactured as described herein, for example usingcomputer-aided design (CAD) and computer-aided manufacture (CAM)methods. However, a person having ordinary skill in the art willappreciate that a variety of other methods may be used to generate andcustomize bioreactors, bioreactor graft chambers, and bioreactor graftchamber frames or inserts according to the present invention.

Size and Shape Variations

As used herein, the terms “corresponding to” and “correspond to,” whenused in relation to any aspect of the present invention where size andshape matching of two or more elements is contemplated, can mean any ofthe size and shape variations described in this section. Such variationsdescribed in this section can apply equally to all aspects of thepresent invention where size and shape matching of two or more elementsis contemplated. Such elements include, tissue portions, tissue models,tissue grafts, model segments, tissue segments, bioreactors, bioreactorchambers (e.g., bioreactor graft chambers) and inserts (e.g., bioreactorgraft chamber inserts), scaffolds, scaffold precursors, cell/scaffoldconstructs, and any other element of the invention as described in thepresent application.

The illustrative embodiments in this section describe size and shapevariations between two elements of the invention—a first element and asecond element. However the present invention contemplates that anydesired number of elements, such as three, four, five or more, may havecorresponding sizes and shapes as described herein. Numerouscombinations of elements are envisioned and are within the scope of thepresent invention, including, but not limited to those describedelsewhere in the present specification and those that combine any one ormore of the elements described above or elsewhere in the application.The variations described in this section apply equally to any suchcombinations where elements may be matched by size and shape.

In some embodiments where a first element has a size and shapecorresponding to a second element, the first element has the same, orabout the same, or approximately the same size and shape as the secondelement. In some embodiments where a first element has a size and shapecorresponding to a second element, the first element has a similar orcomplementary size and shape as the second element.

In some embodiments where a first element has a size and shapecorresponding to the size and shape of a second element, the size andshape of the first element varies by plus or minus 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%,4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%,11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%,17.5%, 18%, 18.5%, 19%, 19.5%, or 20% of the size and shape of thesecond element.

For example, in some embodiments the present invention utilizes athree-dimensional model having a size and shape corresponding to aparticular tissue portion (e.g., a portion of tissue to be constructed,replaced, or repaired). In some embodiments the present inventionutilizes a three-dimensional model segment having a size and shapecorresponding to a cell scaffold, a bioreactor, a graft chamber, a graftchamber insert, and/or a tissue segment. In some embodiments the presentinvention provides a cell scaffold or cell scaffold precursor having asize and shape corresponding to a tissue portion model, a model segment,a bioreactor, a graft chamber, a graft chamber insert, a tissue segment,and/or a tissue graft. In some embodiments the present inventionprovides a bioreactor having a size and shape corresponding to a tissueportion model, a model segment, a scaffold, a graft chamber, a graftchamber insert, a tissue segment, and/or a tissue graft. In someembodiments the present invention provides a bioreactor graft chamber ora bioreactor graft chamber insert having a shape and size correspondingto tissue portion model, a model segment, a tissue segment, and/or atissue graft. In some embodiments the present invention provides atissue segment having a size and shape corresponding to a model segment,a bioreactor, a scaffold, a graft chamber, and/or a graft chamberinsert. In some embodiments the present invention provides a tissuegraft having a size and shape corresponding to a particular tissueportion and/or a three-dimensional model of a particular tissue portion.

Acceptable variations in size and shape can also be determined based onthe desired function of the two or more elements to be matched by sizeand shape. In some embodiments where a first element has a size andshape corresponding to the size and shape of a second element, the firstand second elements can have any suitable size and shape suitable thatallows one or both elements to perform a desired function and/or have adesired property. For example, in some such embodiments a tissue grafthas a size and shape corresponding to a portion of tissue to be repairedprovided that the tissue graft is capable of suitably repairing thetissue portion. In some such embodiments a cell scaffold has a size andshape corresponding to a graft chamber or graft chamber insert providedthat the cell scaffold fits into the graft chamber or graft chamberinsert under press fit conditions.

In addition, a person having ordinary skill in the art will appreciatethat other acceptable variations in size and shape can be determined andthat such variations are intended fall within the scope of the presentinvention.

Three-Dimensional Models

In some embodiments of the present invention, three-dimensional modelsof a particular tissue or tissue portion may be generated and/or used,for example to serve as a template for the production of a tissue graftor tissue graft segment, and/or to serve as a template for theproduction of a scaffold material to be used in the manufacture of sucha tissue graft or tissue graft segment, and/or to serve as a templatefor the production of a bioreactor, bioreactor chamber, or bioreactorchamber insert that could be used in the production of a tissue graft ortissue graft segment (see, e.g., FIGS. 15A-15B). In some embodimentssuch three-dimensional models are digital models, such as digital modelsthat represent the three-dimensional shape and size of a tissue portionof interest. For example, three-dimensional models or images, such asdigital models or images of structures inside the body, can be generatedby any suitable method known in the art, including, for example,computed tomography (CT) (including small-scale CT such as micro-CT)which uses x-rays to make detailed pictures of internal body structuresand organs. In some embodiments medical imaging technologies can be usedto generate a digital model of a desired tissue portion, for example atissue portion comprising a defect, such as a skeletal defect, and thatdigital model can then be used to facilitate the manufacture of a tissuegraft, and/or one or more tissue graft segments, for example by enablingthe production of a scaffold material and/or bioreactor that is customdesigned to be used in the manufacture of the desired tissue graft ortissue graft segment. A model of a tissue portion will preferably beanatomically accurate, having dimensions, geometry, size and shape thatcorrespond to the physical tissue portion and/or the desired tissuegraft. In some embodiments, the portion of tissue may comprise a defect,such as a traumatic or pathological defect. In some embodiments, suchdefect can be repaired using a tissue graft prepared according to thepresent invention. Digital models of tissue portions can be createdusing any suitable computer-aided design (CAD) software, such asAutocad®, Solidworks®, ProE®, or Creo®. In some embodiments a digitalmodel of a tissue portion can be edited and segmented/partitioned intotwo or more smaller sub-parts or segments (which may be referred to as“model segments” or “model portions”), for example representing tissuegraft segments that can be prepared according to the present invention,and/or representing scaffold materials or bioreactor chambers that canbe used for the preparation of such tissue graft segments. The thicknessof the model segments can be selected such that a tissue graft segmenthaving the same thickness could be effectively perfused in a bioreactorof the present invention. Thus, in some embodiments, a model segment,and/or a corresponding tissue graft segment (e.g., a bone graftsegment), has a thickness or a maximum thickness of about one centimeteror less. In some embodiments, the model segment and/or the correspondingtissue graft segment has a thickness or a maximum thickness of about 0.3millimeters to about 10 millimeters, or about 0.3 millimeters to about 5millimeters, or about 0.3 millimeters to about 1 millimeter. In someembodiments, the model segment and/or the corresponding tissue graftsegment has a thickness of about 0.3, about 0.5, about 1, about 1.5,about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5,about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5,about 9, about 9.5, or about 10 millimeters.

The models, such as digital models, described herein can be used todesign and manufacture customized bioreactors as described herein and/orcustomized scaffolds to grow physical tissue graft segments having asize and shape corresponding to the complementary models. In the case ofdigital models, the models or model segments can be created using, orconverted into, any suitable file formats, for example, IGES or SLTformats, and can be created using, or imported into, any suitablecomputer-aided manufacturing (CAM) software, for example, SprutCAM.Manufacture of custom bioreactors and scaffolds is further describedherein.

Digital models of tissues, and segments thereof, provided by theinvention can be generated, edited and otherwise manipulated asdescribed herein. In addition, a person having ordinary skill in the artwill appreciate that any other suitable methods may be used to generate,edit or otherwise manipulate digital models of tissues or segmentsthereof as described herein.

Cell Scaffolds

In some embodiments, the present invention provides scaffolds suitablefor use in the preparation of tissue grafts and/or tissue graftsegments, for example as described herein. Scaffolds can be made of anysuitable material having appropriate pore sizes, porosity and/ormechanical properties for the intended use. In some embodiments cellscaffolds provided by the invention may have a three-dimensionalstructure and can be made of any compliant biomaterial with appropriateporosity, pore size and mechanical properties. Such suitable materialswill typically be non-toxic, biocompatible and/or biodegradable, andcapable of infiltration by cells of the desired tissue graft type, forexample bone-forming cells in the case of bone tissue grafts.Non-limiting examples of such materials include de-cellularized tissue(such as de-cellularized bone), materials that comprise or one or moreextracellular matrix (“ECM”) components such as collagen, laminin,and/or fibrin, and natural or synthetic polymers or composites (such asceramic/polymer composite materials). In some embodiments the scaffoldmaterial may be capable of being absorbed by cells (e.g., resorbablematerials), while in other embodiments non-resorbable scaffold materialsmay be used. In some embodiments, the scaffold may comprise, consist of,or consist essentially of, any of the above-listed materials, or anycombination thereof.

In some embodiments, the dimensions and geometry of a scaffoldcorrespond to that of a three-dimensional model, such as a digitalmodel, of a tissue portion or tissue segment, and/or correspond to thatof the desired tissue graft of tissue graft segment, as described above.In some embodiments the dimensions and geometry of a scaffold can bedesigned or selected based on such a model in order to facilitateculturing of cells, e.g., tissue-forming cells or other cells asdescribed herein, on the scaffold within a bioreactor, as describedherein, for example in order to produce a tissue graft or tissue graftsegment having a size and shape corresponding to a model or modelsegment. In some embodiments, scaffolds may be designed to fit into abioreactor chamber or graft chamber insert of suitable size and shape toallow direct perfusion of the scaffold and the cells therein (e.g.,during the process of producing the tissue graft and/or tissue graftsegment) under press-fit conditions. FIGS. 12A-12B show illustrativescaffolds as provided herein.

In some embodiments cells, for example bone progenitor cells, may beseeded onto the scaffold, then the cell/scaffold structure may be placedor inserted into the graft chamber of a perfusion bioreactor. Thescaffold may be positioned using forceps or the like. In someembodiments, fabricated scaffolds are sterilized and/or conditioned inculture medium prior to cell seeding. In some embodiments cells may growon or within the scaffold under direct perfusion conditions in thebioreactor until maturation of functional tissue, e.g., bone tissue.

In some embodiments, the scaffold is generated or customized usingcomputer-assisted manufacturing (CAM). For example, a tissue modelsegment file can be used with, CAM software to drive the fabrication ofgeometrically defined scaffolds using any suitable method known in theart, or a combination thereof, for example, computer-controlled millingmethods, rapid prototyping methods, laser cutting methods,three-dimensional printing, and/or casting technologies. In someembodiments, manufacturing of the scaffold comprises using rapidprototyping, a milling machine, casting technologies, laser cutting,and/or three-dimensional printing, or any combination thereof. In someembodiments, manufacturing of the scaffold comprises usingcomputer-numerical-control, such as when the manufacturing compriseslaser cutting or using a milling machine. For example, digital models,such as those generated using CAD software as described above, can beprocessed to generate the appropriate codes (such as “G-Codes”) to drivea computer-numerical-control (CNC) milling machine (for example,Tormach, Bridgeport) and to select appropriate machining tool bits andprogram machining paths to cut the scaffold material into the desiredshapes and sizes (e.g., corresponding to that of a digital models of atissue segment).

While scaffolds provided by the invention can be designed andmanufactured as described herein, a person having ordinary skill in theart will appreciate that a variety of other methods of designing andmanufacturing may be used to generate scaffolds according to the presentinvention.

Cells

Any suitable or desired type of cell or cells may be used in thepreparation of tissue grafts or tissue graft segments in accordance withthe present invention, as described herein. Typically the selectedcell(s) will be capable of forming the desired tissue graft (forexample, for a vascularized bone graft, mesenchymal progenitor cells andendothelial progenitor cells or any other cell types suitable for orcapable of forming bone and blood vessels, as further described herein),or any cell(s) capable of differentiating into the desiredtissue-forming cell(s) (for example, a pluripotent cell). Non-limitingexamples of cells that may be used include pluripotent cells, stemcells, embryonic stem cells, induced pluripotent stem cells, progenitorcells, tissue-forming cells, or differentiated cells.

The cells used may be obtained from any suitable source. In someembodiments, the cells may be human cells. In some embodiments, thecells may be mammalian cells, including, but not limited to, cells froma non-human primate, sheep, or rodent (such as a rat or mouse). Forexample, cells may be obtained from tissue banks, cell banks or humansubjects. In some embodiments, the cells are autologous cells, forexample, cells obtained from the subject into which the prepared tissuegraft will be subsequently transplanted, or the cells may be derivedfrom such autologous cells. In some embodiments, the cells may beobtained from a “matched” donor, or the cells may be derived from cellsobtained from a “matched” donor. For cell and tissue transplants, donorand recipient cells can be matched by methods well known in the art. Forexample, human leukocyte antigen (HLA) typing is widely used to match atissue or cell donor with a recipient to reduce the risk of transplantrejection. HLA is a protein marker found on most cells in the body, andis used by the immune system to detect cells that belong in the body andcells that do not. HLA matching increases the likelihood of a successfultransplant because the recipient is less likely to identify thetransplant as foreign. Thus, in some embodiments of the presentinvention, the cells used are HLA-matched cells or cells derived fromHLA-matched cells, for example, cells obtained from a donor subject thathas been HLA-matched to the recipient subject who will receive thetissue graft. In some embodiments the cells used may be cells that havebeen modified to avoid recognition by the recipient's immune system(e.g., universal cells). In some such embodiments the cells aregenetically-modified universal cells. For example, in some embodimentsthe universal cells may be MHC universal cells, such as majorhistocompatibility complex (MHC) class I-silenced cells. Human MHCproteins are referred to as HLA because they were first discovered inleukocytes. Universal cells have the potential to be used in anyrecipient, thus circumventing the need for matched cells.

In some embodiments, the cells used in making the tissue graftsdescribed herein, include pluripotent stem cells, such as inducedpluripotent stem cells (iPSCs). In some such embodiments, thepluripotent stem cells may be generated from cells obtained from thesubject (i.e., autologous cells) that will receive the tissue graft. Inother such embodiments, the pluripotent stem cells may be generated fromcells obtained from a different individual, i.e., not the subject thatwill receive the tissue graft (i.e., allogeneic cells). In some suchembodiments, the pluripotent stem cells may be generated from cellsobtained from a different individual, i.e., not the subject that willreceive the tissue graft, but where that different individual is a“matched” donor, for example as described above. In some embodiments,the cells used are differentiated cells, such as bone cells. In someembodiments, the differentiated cells are derived from pluripotent stemcells, such as induced pluripotent stem cells. In some embodiments, thedifferentiated cells are derived by trans-differentiation ofdifferentiated somatic cells, or by trans-differentiation of pluripotentcells (such as pluripotent stem cells or induced pluripotent stemcells), for example induced pluripotent stem cells generated fromsomatic cells.

A pluripotent stem cell is a cell that can (a) self-renew and (b)differentiate to produce cells of all three germ layers (i.e., ectoderm,mesoderm, and endoderm). The term “induced pluripotent stem cell”encompasses pluripotent stem cells, that, like embryonic stem cells(ESC), can be cultured over a long period of time while maintaining theability to differentiate into cells of all three germ layers, but that,unlike ES cells (which are derived from the inner cell mass ofblastocysts), are derived from somatic cells, that is, cells that had anarrower, more defined potential and that in the absence of experimentalmanipulation could not give rise to cells of all three germ layers.iPSCs generally have an hESC-like morphology, growing as flat colonieswith large nucleo-cytoplasmic ratios, defined borders and prominentnuclei. In addition, iPSCs generally express one or more keypluripotency markers known by one of ordinary skill in the art,including but not limited to Alkaline Phosphatase, SSEA3, SSEA4, Sox2,Oct3/4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26a1,TERT, and zfp42. In addition, iPSCs, like other pluripotent stem cells,are generally capable of forming teratomas. In addition, they aregenerally capable of forming or contributing to ectoderm, mesoderm, orendoderm tissues in a living organism.

Illustrative iPSCs include cells into which the genes Oct-4, Sox-2,c-Myc, and Klf have been transduced. Other exemplary iPSCs are cellsinto which OCT4, SOX2, NANOG, and LIN28 have been transduced. One ofskill in the art would know that various different cocktails ofreprogramming factors can be used to produce iPSCs, such as factorsselected from the group consisting of OCT4, SOX2, KLF4, MYC, Nanog, andLin28. The methods described herein for producing iPSCs are illustrativeonly and are not intended to be limiting. Rather any suitable methods orcocktails of reprogramming factors known in the art can be used. Inembodiments where reprogramming factors are used, such factors can bedelivered using any suitable means known in the art. For example, insome embodiments any suitable vector, such as a Sendai virus vector, maybe used. In some embodiments reprogramming factors may be deliveredusing modified RNA methods and systems. A variety of different methodsand systems are known in the art for delivery of reprogramming factorsand any such method or system can be used.

Any culture medium suitable for culture of cells, such as pluripotentstem cells, may be used in accordance with the present invention, andseveral such media are known in the art. For example, a culture mediumfor culture of pluripotent stem cells may comprise Knockout DMEM, 20%Knockout Serum Replacement, nonessential amino acids, 2.5% FBS,Glutamax, beta-mercaptoethanol, 10 ng/microliter bFGF, and antibiotic.The employed medium may also be a variation of this medium, for examplewithout the 2.5% FBS, or with a higher or lower % of knockout serumreplacement, or without antibiotic. The employed medium may also be anyother suitable medium that supports the growth of human pluripotent stemcells in undifferentiated conditions, such as mTeSR (available fromSTEMCELL Technologies), or Nutristem (available from Stemgent), or ESmedium, or any other suitable medium known in the art. Other exemplarymethods for generating/obtaining pluripotent stem cells from apopulation of cells obtained from a subject are provided in the Examplesof the present application.

In some embodiments, pluripotent stem cells are differentiated into adesired cell type, for example, a bone-forming cell or a bloodvessel-forming cell, or any other desired cell type. Differentiatedcells provided by the invention can be derived by various methods knownin the art using, for example, adult stem cells, embryonic stem cells(ESCs), epiblast stem cells (EpiSCs), and/or induced pluripotent stemcells (iPSCs; somatic cells that have been reprogrammed to a pluripotentstate). Methods are known in the art for directed differentiation orspontaneous differentiation of pluripotent stem cells, for example byuse of various differentiation factors. Differentiation of pluripotentstem cells may be monitored by a variety of methods known in the art.Changes in a parameter between a stem cell and a differentiationfactor-treated cell may indicate that the treated cell hasdifferentiated. Microscopy may be used to directly monitor morphology ofthe cells during differentiation.

In each of the embodiments of the invention, any suitable or desiredtypes of cells can be used to produce the tissue grafts and tissue graftsegments described herein, including, but not limited to, pluripotentstem cells or progenitor cells or differentiated cells. In someembodiments, the pluripotent stem cells may be induced pluripotent stemcells. In embodiments where induced pluripotent stem cells are used,such cells may be derived from differentiated somatic cells obtainedfrom a subject, for example by contacting such differentiated somaticcells with one or more reprogramming factors. In some embodiments,pluripotent cells may have been induced toward a desired lineage, forexample, mesenchymal lineage or endothelial lineage. In someembodiments, the differentiated cells can be any suitable type ofdifferentiated cells. In some embodiments, the differentiated cells maybe derived from pluripotent stem cells (such as induced pluripotent stemcells), for example by contacting such pluripotent cells with one ormore differentiation factors. In some embodiments, the differentiatedcells may be derived by trans-differentiation of another differentiatedcell type, for example by contacting the cells with one or morereprogramming factors. In the various embodiments of the presentinvention involving differentiated cells, such differentiated cells maybe any desired differentiated cell type, including, but not limited to,bone cells and blood vessel cells.

Cell/Scaffold Constructs

Any suitable or desired type of cell, such as the cell types describedherein, can be applied to or seeded onto a scaffold to prepare tissuegraft or tissue graft segment according to the present invention.

In some embodiments, cells may be in a differentiated state prior tobeing applied to a scaffold. For example, in some embodimentsdifferentiated cells may be obtained and used directly. Similarly, insome embodiments non-differentiated cells may be cultured according toany suitable method known in the art, such as in a culture dish ormulti-well plate or in suspension, for a suitable period or length oftime, for example, until desired levels of cell growth ordifferentiation or other parameters are achieved, then thedifferentiated cells may be transferred to the scaffold and subsequentlythe cell/scaffold construct is inserted into a bioreactor to facilitatedevelopment of a tissue graft or tissue graft segment. In someembodiments, non-differentiated cells (for example, stem cells (such asiPSCs) or progenitor cells) may be applied to the scaffold. In suchembodiments, the non-differentiated cells may undergo differentiationwhile being cultured on the scaffold.

In some embodiments, two or more different cell populations may beseeded onto a scaffold to prepare a cell/scaffold construct. Forexample, in some embodiments both bone-forming cells and bloodvessel-forming cells may be seeded onto a scaffold and co-cultured forthe preparation of a vascularized bone graft. In some embodiments, thetwo or more populations of cells are co-cultured on the scaffold for asuitable period of time, for example, until desired levels of growth ordifferentiation or other parameters are achieved, before thecell/scaffold construct is inserted into the bioreactor. Populations ofcells may comprise, consist essentially of, or consist of, any desiredtype of cell in any stage of growth or differentiation, and anycombinations thereof. For example, in some embodiments, each cellpopulation may comprise cells capable of forming a different tissue, forexample for the preparation of a vascularized bone graft, a firstpopulation containing cells capable of forming bone, such as mesenchymalprogenitor cells, and a second population containing cells capable offorming blood vessels, such as endothelial progenitor cells. In someembodiments, each population of cells may comprise cells capable offorming the same tissue (e.g., bone) but each population of cells may beat different stages of differentiation (e.g., mesenchymal stem cells andbone marrow stromal cells). Populations of cells to be co-cultured maybe applied to a scaffold at the same time or at different times, asdesired. Where two or more populations of cells are applied at differenttimes, the sequence or order of co-culture (e.g., which population isapplied to the scaffold first, which population is applied to thescaffold second, etc.) may be selected as desired, for example dependingon the cell types being used, the state or growth or differentiation ofthe populations of cells, or any other parameters, as desired. Where twoor more populations of cells are to be applied to the scaffold, they canbe applied at any suitable cell ratio, as desired. For example, in someembodiments two different populations of cells may be seeded at a ratioof about 1:1, or any ratio from about 2:8 to about 8:2. In someembodiments, the cell populations may be seeded at a ratio of about 2:8,about 3:7, about 4:6, about 5:5, about 6:4, about 7:3, or about 8:2.

A cell/scaffold construct may be transferred to a bioreactor of thepresent invention at any suitable point, for example, immediately afterseeding with cells, following a certain period of cell culture followingseeding, after the seeded cells have reached a desired state ofdifferentiation or any other desired state, as desired. In someembodiments the cell/scaffold construct is inserted into a bioreactorand cultured under press fit conditions to allow formation of a tissuegraft or tissue graft segment. Tissue/graft development can be assessedusing any suitable qualitative or quantitative methods known in the art,including but not limited to histological and immunohistochemicalexamination, biochemical assays, high-resolution characterizationtechniques (e.g., SEM, FIB-TEM, Tof-SIMS), imaging procedures (e.g., CTor microCT) and mechanical testing (e.g., Young's modulus, tensile andcompressive strength).

A person having ordinary skill in the art will recognize that countlessvariations and combinations of cells and culture methods will fallwithin the scope of the present invention. For example, cell culturemethods, including cell seeding ratios, concentration of differentiationfactors and sequence of co-culture, will typically be determinedaccording to the desired cell type being used or the tissue graft beingprepared.

Tissue Grafts, and Assembly and Use Thereof

In some embodiments, the present invention provides tissue grafts, suchas bone grafts, that are assembled from multiple tissue graft segmentsgenerated in a bioreactor of the present invention. The presentinvention also provides methods of making such tissue grafts. Suchmethods may be referred to as segmental additive tissue engineering(SATE) methods. In the case of bone grafts in particular, such methodsmay be referred to as segmental additive bone engineering (SABE)methods. At any suitable point, for example when a tissue graft segmenthaving the desired properties has been produced, tissue graft segmentscan be removed from the bioreactor in which they are produced andmultiple tissue graft segments can be assembled together to form atissue graft having the desired size and shape, for example a size andshape corresponding to the tissue portion to be replaced.

Assembled tissue graft segments can be secured or attached together byany suitable means or method capable of maintaining the intendedassembly of the segments. For example typically, such securing means ormethods will be non-toxic, biocompatible and/or resorbable (e.g.,capable of being absorbed by the body), for example, where the assembledtissue graft will be transplanted into a subject. For example, in someembodiments, the tissue graft segments may be secured to each otherusing an adhesive, stitches or sutures, staples, plates, pins and holes,screws, bolts, or the like, as desired. In some embodiments, the meansused to secure the tissue segments together are biocompatible orresorbable or both.

In some embodiments, where an adhesive is used to secure the graftsegments to each other, the adhesive may be a biocompatible glue, forexample, a biocompatible polymer glue such as NovoSorb® (PolyNovoBiomaterials, Melbourne) or any gel, liquid, rubber-like substance, orother biocompatible adhesive material capable of securing together twoor more tissue graft segments. For example, in the case of bone grafts,exemplary bone glues that can be used to secure bone graft segments toeach other include, but are not limited to, polymer-based or polymericbone glues such as polyurethane-based and polymethylmethacrylate-basedbone glues. In some embodiments, the adhesive may be a tape, forexample, a surgical tape. In some embodiments, tissue graft segments maybe secured to each other using one or more plates, pins, screws, bolts,staples, stitches, sutures, or the like, for example made of plastic,metal (for example, titanium) or any other suitable material. In someembodiments such pins, screws, bolts, staples, stitches, sutures, or thelike may be manufactured using 3D printing or any other suitable methodknown in the art.

In some embodiments, various different means and/or methods to securethe assembled tissue graft segments together may be used in combination,for example, to reinforce the connection between the assembled tissuegraft segments and/or to attach or anchor or secure the tissue graft tothe host tissues, such as where a tissue graft is transplanted into asubject. For example, in some embodiments engineered bone graft segmentsas described herein can be assembled together using both a biocompatiblebone glue and metallic or resorbable pins.

Following assembly and securing together of the tissue graft segments,the resulting tissue graft can be transplanted into a subject, where itmay also be anchored to the subject's tissues (such as surrounding bonein the case of a bone graft). In some embodiments, the methods andcompositions provided by the present invention may be used to engineertissue grafts for clinical applications, including therapeutic and/orcosmetic applications. Non-limiting examples of such applicationsinclude repair or replacement of a tissue defect or damage or tissueloss, tissue reconstruction or rebuilding, tissue reinforcement (e.g.,to prevent or delay progression of tissue damage or loss of tissue) orto assist in the implantation of surgical devices (e.g., bone grafts canbe used to help bone heal around surgically implanted devices such asjoint replacements, plates or screws). In some embodiments, a subjecthas a tissue defect or tissue loss caused by injury, disease, birthdefect, trauma or infection.

In some embodiments, the invention provides a method of repairing orreplacing a tissue defect, tissue loss or tissue damage, comprisingtransplanting a tissue graft according to the present invention into asubject so as to repair or replace the tissue defect, tissue loss ortissue damage in the subject. In some embodiments, the tissue graft willhave a size and shape corresponding to that of the tissue being repairedor replaced. Tissue grafts according to the present invention can beprepared using the segmental additive tissue engineering or SATE methodsprovided herein. Thus, in some embodiments, a tissue graft according tothe present invention may comprise, consist of, or consist essentiallyof, two or more tissue graft segments, wherein the tissue graft segmentshave a thickness of less than about 1 centimeter, or a thickness ofabout 0.3 millimeters to about 10 millimeters. In some embodiments, sucha tissue graft may be an autograft (also referred to as an autogenous,autogenic or autogenic graft), such as where the subject's own cells ortissue (e.g., autologous cells or tissue) are used to generate thetissue graft. In some embodiments, the tissue graft is an allograft(e.g., the tissue graft is generated from cells or tissues obtained froma donor subject of the same species as the recipient subject), such aswhere the donor and recipient subjects have been matched, for example,by HLA-matching. In some embodiments, the tissue graft is a xenograft(e.g., the tissue graft is generated from cells or tissues obtained froma donor subject of a different species as the recipient subject). Forexample, a tissue graft comprising human tissue may be transplanted intoa non-human mammal, such as a sheep, for example for performing certainin vivo testing, etc.

A tissue graft prepared according to the present invention andtransplanted into a subject can be anchored or attached or secured toexisting structures (e.g., tissue) in the subject by any suitable methodcapable of securing tissue, such as described above. In someembodiments, the transplanted tissue graft is secured by an adhesive,stitches or sutures, staples, plates, pins or the like. In someembodiments, the means to secure the tissue graft inside the subject'sbody will be biocompatible or resorbable or both.

Subjects

In some embodiments the cells used in producing the tissue grafts of thepresent invention may be obtained from or derived from any subject, asneeded or as desired. In some embodiments the methods (e.g., treatmentmethods) and compositions (e.g., tissue grafts) provided by the presentinvention may be used in any subject, as needed or as desired (forexample, to repair a pathological or traumatic tissue defect, or forcosmetic or reconstructive purposes). In some embodiments, the subjectis a human. In some embodiments, the subject is a mammal including butnot limited to a non-human primate, sheep, or rodent (such as a rat ormouse). In some embodiments, a first subject is a donor subject and asecond subject is a recipient subject. In some such embodiments thedonor subject, or cells of the donor subject, may be matched to therecipient subject or cells of the recipient subject, for example, byHLA-type matching.

Model Systems and Screening Methods

In some embodiments, the present invention provides model systems forstudying various biological processes or biological properties, andscreening methods for testing the effects of various agents on suchbiological processes and/or biological properties.

In some embodiments, the present invention provides a model systemcomprising a tissue graft according to the present invention. Forexample, in some embodiments the present invention provides a modelsystem comprising a tissue graft according to the present invention thathas been implanted into a subject. Model systems provided by theinvention can be used for various purposes such as but not limited toscreening or testing materials for implantation and to study diseasesunder defined tissue-specific conditions, including for understandingunderlying mechanisms, defining therapeutic targets and conductingcompound screening, and the like.

Furthermore, those of ordinary skill in the art will appreciate that themethods, compositions (e.g., tissue grafts), and devices (e.g.,bioreactors), described herein can be used in, or in conjunction with, avariety of different model systems and screening methods.

Vascularized Bone Grafts

In one embodiment, the present invention provides a method of preparinga vascularized bone graft, comprising: (a) obtaining a three-dimensionalmodel of a bone portion; (b) partitioning the three-dimensional model ofstep (a) into two or more bone segment models; (c) preparing two or morebone graft segments, comprising: (i) obtaining a scaffold having a sizeand shape corresponding to each of the bone segment models of step (b);(ii) obtaining a bioreactor having an internal chamber configured tohold the scaffold; (iii) applying to the scaffold (1) bone-formingcells, or cells capable of differentiating into bone-forming cells, and(2) blood vessel-forming cells, or cells capable of differentiating intoblood-vessel forming cells; (iv) culturing the cells on the scaffoldwithin the bioreactor to form a bone graft segment; and (v) removing thebone graft segment from the bioreactor; and (d) assembling the two ormore bone graft segments prepared in step (c) to form a bone grafthaving a size and shape corresponding to the bone portion of step (a).In one embodiment, the cells applied to the scaffold in (c) (iii)comprise pluripotent cells, induced pluripotent cells, progenitor cells,differentiated cells, or any combination thereof. In one embodiment, thecells of (c) (iii) (1) comprise bone marrow stromal cells or mesenchymalstem cells or pluripotent cells induced toward mesenchymal lineage ordifferentiated bone cells or any combination thereof. In one embodiment,the cells of (c) (iii) (2) comprise endothelial progenitor cells orpluripotent cells induced toward endothelial lineage or differentiatedendothelial cells or any combination thereof. In one embodiment, thebone graft segment has a thickness of about one centimeter or less. Inone embodiment, the bone graft segment has a thickness of about 0.3millimeters to about 10 millimeters. In one embodiment, the assemblingof the bone graft segments is carried out with an adhesive, one or morepins and holes, or both. In one embodiment, the pins are metallic orresorbable. In one embodiment, the pins are titanium. In one embodiment,the adhesive is a biocompatible bone glue, for example, a polymer suchas NovoSorb® (PolyNovo Biomaterials, Melbourne) or any gel, liquid,rubber-like substance or any other biocompatible material capable ofsecuring together two or more bone segments. Examples of bone gluesinclude, but are not limited to, polymer based bone glues such aspolyurethane-based and polymethylmethacrylate-based bone glues. In oneaspect, the invention provides a method of repairing or replacing a boneportion in a subject, comprising steps (a)-(d) described above, andfurther comprising transplanting the bone graft into a subject so as torepair or replace the bone portion in the subject.

In one aspect, the invention provides a vascularized bone graft preparedby a method of the invention utilizing a bioreactor as described herein.In another aspect, the invention provides a vascularized bone graft forrepairing or replacing a bone portion in a subject, wherein the bonegraft comprises two or more bone graft segments, wherein the two or morebone graft segments are connected together to form a vascularized bonegraft having a size and shape corresponding to the bone portion to bereplaced or repaired. In some embodiments, the bone graft segmentscomprise bone cells derived from progenitor cells (such as mesenchymalprogenitor cells), pluripotent cells (such as induced pluripotent stemcells), autologous cells (such as the subject's own cells), or any cellcapable of (i) forming bone, or (ii) differentiating into a cell that iscapable of forming bone. In some embodiments, the bone graft segmentscomprise endothelial or blood vessel cells derived from progenitor cells(such as endothelial progenitor cells), pluripotent cells (such asinduced pluripotent stem cells), autologous cells (such as the subject'sown cells), or any cell capable of (i) forming endothelium and/or bloodvessels, or (ii) differentiating into a cell that is capable of formingendothelium and/or blood vessels. In some embodiments, each bone segmenthas a maximum thickness of less than about one centimeter, or has amaximum thickness of about 0.3 millimeters to about 10 millimeters.

In some embodiments the cells used in accordance with the above methods,or used in the manufacture of the above bone grafts, are derived from,or derived from a cell obtained from, the same subject into which thebone graft is to be placed such that they are autologous cells, or arederived from autologous cells. In one embodiment, the cells are derivedfrom pluripotent stem cells, such as, for example, induced pluripotentstem cells, embryonic stem cells, cloned stem cells, or adult stem cells(such as bone marrow stem cells). In some embodiments the inducedpluripotent stem cells may be derived from a somatic cell taken from thesame subject into which the bone graft is to be placed or from asuitably matched donor, such as HLA-matched. In some embodiments, thecells are mesenchymal stem cells and/or endothelial progenitor cells. Insome embodiments, the cells are seeded onto the scaffold at a cell ratioof 1:1, or any ratio from about 2:8 to about 8:2.

The following examples are provided to further illustrate the advantagesand features of the present invention, but are not intended to limit thescope of the invention. While they are typical of those that might beused, other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used.

EXAMPLES Example 1 Use of Bioreactors and Scaffolds to Engineer LargeBone Grafts

Computed tomography is used to generate anatomically accurate 3D modelsof traumatic or pathological skeletal defects. Digital models ofskeletal defects are then edited using computer-aided design (CAD), andsliced off into pieces of adequate thickness, which allow effectiveengineering of functional bone tissue using perfusion bioreactors. Thepartitioned bone defect files are thus imported in computer-aidedmanufacture (CAM) software to drive the fabrication of geometricallydefined biomaterial scaffolds using a combination of computer-controlledmilling, rapid prototyping and casting technologies. The partitionedbone defect files are also used to drive the computer-controlled millingor 3D printing of customized perfusion bioreactors that can accommodatethe cell/scaffold construct in a press-fit fashion under directperfusion conditions (see FIG. 14, top panel). Patient-specific bone andblood vessel forming cells are then seeded onto the produced scaffolds,and the cell/scaffold constructs cultured in the relative perfusionbioreactor chambers until maturation of functional vascularized bonetissue (see FIG. 14, middle panel). Engineered bone segments are thenassembled to match the shape of the skeletal defect by means of abiocompatible bone glue for welding large bone grafts, and/or reinforcedusing 3D printed metallic or resorbable pins that integrate and create astable bond with bone. Following assembling of the segments, theengineered bone grafts is anchored to the host tissues using a similarapproach based on biocompatible bone glue and pins resulting inpersonalized reconstruction of skeletal defects (see FIG. 14, bottompanel).

Example 2 Computer-Aided Design and Manufacture of Bioreactors andScaffolds

Bioreactors and scaffolds provided by the invention can be designed andmanufactured as described herein. In some embodiments, computer-aideddesign (CAD) and computer-aided manufacture (CAM) are used to design andmanufacture the bioreactors and/or scaffolds. A person having ordinaryskill in the art will appreciate that any other methods of designing andmanufacturing may be used to generate bioreactors and/or scaffoldsaccording to the present invention.

Digital models of skeletal defects can be created using CAD software,e.g., Autocad, Solidworks, ProE, and Creo. Reference models of skeletaldefects in CAD can be edited and segmented into smaller complementarysub-parts which represent bone segments that can be cultured inperfusion bioreactors without affecting the perfusion system. Thesegmented bone sample files can be saved in compatible formats, forexample, IGES or SLT formats, and imported in CAM software (e.g.,SprutCAM).

The segmented bone sample files edited in CAD can be used to design acustomized bioreactor, which is of suitable size and shape toaccommodate a scaffold construct in a press-fit fashion under directperfusion conditions. The CAD files are converted into compatibleformats and imported into CAM and/or 3D printer software, and used tofabricate the bioreactors using different plastic materials.

The generated files in CAM software can be processed to generate theappropriate G-Codes to drive a computer-numerical-control (CNC) millingmachine (for example, Tormach, Bridgeport) and to select appropriatemachining tools bits and program the machining paths to cut thescaffolding materials into the desired segmented shapes. For example,plugs of trabecular bone (cow and/or human) of adequate size can bedrilled, cleansed under high-pressure streamed water to remove the bonemarrow, and then sequentially washed to remove cellular material.Decellularized bone plugs will then be freeze-dried, and used for thefabrication of scaffolds corresponding to the shape and size of thesegmented samples of the skeletal defect. The invention providesscaffolds made of synthetic, resorbable and/or mechanically compliantceramic/polymer composite materials. The efficient manufacture ofscaffolds is important for the reproducible and large-scale fabricationof bone substitutes for clinical applications.

Example 3 Engineering Vascularized Bone Grafts for Repairing LargeSkeletal Defects INTRODUCTION

This Example proposes a strategy for engineering vascularized bonegrafts from human induced pluripotent stem cells (hiPSCs) for enhancedhealing of complex skeletal defects. In particular, the ability toderive autologous osteogenic and vascular cells constituting healthybone from hiPSCs for any patient in virtually unlimited numbersrepresents an unprecedented therapeutic resource. Vascularized bonesubstitutes will be engineered using a biomimetic scaffold-bioreactorapproach to bone development. Computer-aided and rapid prototypingtechnologies will allow the preparation of bone substitutes of any shapeand size. Digital models of large bone defects will be created and thensegmented in complementary sub-parts that will be used to producecustomized biomaterial scaffolds and bioreactors via computer-aideddesign and manufacturing technologies, such as 3D printing (see FIG.14). The proposed engineering strategy overcomes the limitationsassociated with perfusion culture of large bone grafts. Mesenchymal andendothelial progenitor cells will be derived from hiPSCs generated usingany available reprogramming method, and then combined with compliantscaffolds and cultured in perfusion bioreactors until maturation offunctional vascularized tissue (see FIG. 14). Engineered bone segmentswill then be assembled together (lego-like approach) using abiocompatible bone glue, and/or reinforced using 3D printed titaniumholes and pins to match the shape and dimension of the original defect.Future studies will be aimed at exploring the therapeutic potential ofhiPSC-engineered bone using different animal models of complex skeletaldefects (see FIG. 14).

Engineering large and geometrically defined vascularized bone graftsfrom hiPSCs represents a novel solution for the treatment of skeletaldefects characterized by severe bone loss, and opens the opportunity toprovide personalized therapies to a large number of patients. Asimportantly, such bone grafts represent qualified models to study bonedevelopment and pathologies, as well as screening new drugs and testbiomaterials.

This Example describes studies designed to engineer vascularized bonegrafts from human induced pluripotent stem cells (hiPSC) for enhancedhealing of skeletal defects. Patient-specific bone grafts will beengineered using a biomimetic scaffold-bioreactor approach of bonedevelopment in vitro, and customized to meet specific clinical needswith the aid of computer-assisted and rapid prototyping technologies.Engineering patient-specific customized bone grafts could be used todevelop innovative treatments to restore skeletal integrity andfunctionality in clinical situations characterized by severe bone loss.

This Example proposes studies to engineer vascularized bone substitutesfrom hiPSCs, and adopt a combination of medical imaging procedures,computer-aided technologies and rapid prototyping to allow theconstruction of clinically relevant bone substitutes in perfusionbioreactors of the present invention. The strategy represents a noveland innovative solution to cope with the burden of bone deficiencies,whose clinical translation will have profound social impact by improvingthe health status and quality of life of many patients. These studieswill also provide new insights into hiPSC biology, which are critical tounderstand functional differentiation of pluripotent stem cells intomature tissues and organs. Additionally, hiPSC-engineered vascularizedbone grafts would provide valuable high-fidelity models to investigatetissue development in normal and pathological conditions, and test newpharmaceuticals and biomaterials within a context that resembles severalaspects of the native bone environment.

Background

Bone displays intrinsic capacity to regenerate and self-repair but thisability is limited to small fractures and reconstructive therapies areneeded in a large number of clinical conditions to restore tissueintegrity and functionality. Current treatments are based on thetransplantation of autogenic and/or allogeneic bone grafts, orimplantation of graft materials with osteoconductive and osteoinductiveproperties. Autogenic bone grafts represent the gold standard treatmentfor bone replacement procedures, due to immune tolerability andprovision of essential components supporting bone regeneration andrepair, but limited availability and donor site morbidity often restricttheir clinical use. On the other hand, allogeneic decellularized bonegrafts are available in large amounts but integrate slowly, carry therisk of infection transmission and may display immune incompatibilityleading to transplant rejection. Implantation of alloplastic materialsovercomes some of the restrictions encountered with autogenic andallogeneic grafts, including disease transmission, complex shape andavailability, but display poor integration, frequently result inbiomaterial-associated infection, and lack biological functionality andmechanical compliance, leading to implant failure and substitution. Bonetissue engineering represents a promising therapeutic solution, since itopens the possibility to engineer an unlimited amount of viable bonesubstitutes to meet specific clinical needs. Human mesenchymal stemcells (hMSC) derived from adult tissues have been extensively used forbone engineering applications with encouraging results, but exhibitrestricted potential for clinical applications due to limitedavailability, inadequate regenerative potential and decrease infunctionality associated with in vitro expansion and donor age.

Autologous bone substitutes in the size range of ˜1 cm have been grownfrom adult stem cells and used to facilitate bone healing inexperimental animals and in humans. However, their scale-up to clinicalsizes and functionality are limited due to the lack of blood supply, andlimited proliferation and vasculogenic potential of cultured adult stemcells. An appropriate blood supply has been recognized as an essentialcomponent of normal fracture healing and defective angiogenesis at thefracture site has been a primary consideration when poor outcomes occur.Poor blood supply leads to hypoxia and necrosis of the grafted tissue,and can result in decreased bone formation (“atrophic bone”). Similarly,implantation of large cellularized bone substitutes without theconnection to vascular supply can result in cell death in the interiorregions of the transplant. To expedite cell survival and boneregeneration, recent tissue engineering approaches have involvedtransplantation of endothelial progenitors or vascular networks withinbone substitutes. Studies have shown the positive effects of endothelialcells and osteogenic cells in direct co-culture model. In addition,studies suggest that co-transplantation of endothelial cells and BMSCpromoted new bone formation in vivo, and that endothelial networksengineered within bone substitutes can functionally anastomose with thehost vasculature.

Pluripotent stem cells display high regenerative potential and abilityto differentiate toward all specialized cells constituting healthy bonetissue. When derived using nuclear reprogramming technologies,pluripotent stem cells allow the construction of patient-specific bonesubstitutes for personalized applications. Both mesenchymal andendothelial progenitor cells have recently been derived from pluripotentstem cells, opening new opportunities for the unlimited construction ofvascularized bone substitutes for enhanced reconstructions of largeskeletal defect. It is therefore important to explore the possibility toengineer vascularized bone grafts from induced pluripotent stem cells,in order to develop safe and effective treatments for many patientsaffected by severe skeletal defects and bone disorders.

Results

The inventors have extensive experience with cultivation of bonesubstitutes from mesenchymal stem cells derived from adult tissues andfrom human pluripotent stem cells. A set of studies exploring therelative regenerative potential of hMSCs and mesenchymal progenitorsderived from human embryonic stem cell (hESC) lines have demonstratedcomparative advantages of hESC-derived mesenchymal progenitors for boneengineering applications. Studies in monolayer and 3D cultures onscaffolds in bioreactors have shown that hESC-derived mesenchymalprogenitors highly resemble hMSCs in terms of morphology, surfaceantigen and global gene expression profile, but display higherproliferation potential, biosynthetic activity and mineralizationproperties, all paramount features for the unlimited construction offunctional substitutes for bone engineering applications. The derivationprotocol has been extended to hiPSC lines generated from differenttissues and using different reprogramming technologies based onnon-integrating vectors, opening the possibility to engineer safepatient-specific bone substitutes for personalized applications. hiPSClines were characterized by immunohistochemistry to assess pluripotencyand karyotyped, before being induced toward the mesenchymal lineage for7 days. Mesenchymal-like phenotype was characterized by flow cytometryand by probing surface marker expression and differentiation potentialin monolayer (osteogenesis, adipogenesis) and pellet cultures(chondrogenesis). Differentiation toward the osteogenic lineage wasconfirmed by alkaline phosphatase and mineralization, differentiationtoward the chondrogenic lineage was shown by glycosaminoglycans, anddifferentiation toward the adipogenic lineages was shown by lipidcharacterization.

Cells were then seeded on decellularized bone scaffolds (4 mm Ø×4 mmheight), and cultured in osteogenic medium under constant perfusion(linear flow velocity of 800 μm/sec) for 5 weeks before 12-weeksubcutaneous implantation in immunocompromised mice to assess stabilityand further tissue maturation. Histological and immunohistochemicalanalyses of engineered bone were carried out following bioreactorcultivation and subcutaneous implantation in immunocompromised mice.Micrographs showed maturation of phenotypically stable bone-like tissueand vascularization. MicroCT analysis of engineered bone showed anincrease in mineral density and structural parameters.

Altogether the results demonstrate that mesenchymal progenitors can bederived from hiPSC lines, and used to engineer mature and phenotypicallystable bone tissue for repair treatments of skeletal defects inpersonalized applications. In all studies, perfusion bioreactors wereshown to be particularly important for bone development, as they providebiomechanical stimulation to the cells, and support survival of thecells in the interior of the constructs, resulting in the production ofthick homogenous bone-like matrix. Studies are now directed atdeveloping suitable protocols for engineering vascularized bonesubstitutes for enhanced healing of large and geometrically complexskeletal defects. Preliminary studies have shown that functionalendothelial progenitors can be derived from hESC lines. Followingdifferentiation of embryoid bodies in controlled conditions, isolatedCD34 positive cells were able to specifically internalize DiI-Ac-LDL andform tubes when plated on Matrigel. This approach is being translated tohiPSCs for the construction of patient-specific multicellular compositebone substitutes.

In addition, preliminary vascularization studies in 3D cultures haveshown that co-culture of hiPSC-derived mesenchymal progenitors and humanbone marrow stromal cells (BMSC) with human umbilical vein endothelialcells (HUVEC) result in long-lasting formation of vascular networks,both when cells are embedded in fibrin clots or seeded ontodecellularized bone scaffolds, which represent more compliant substratesfor skeletal repair treatments. Interestingly, number and stability ofvascular structures were similar when HUVEC were cultured withhiPSC-derived mesenchymal progenitors and human BMSC in fibrin clots.Epifluorescence micrographs showed the presence of stable 3D vascularnetworks 3 weeks after seeding. Hematoxylin/Eosin staining of clot crosssections showed the presence of hollow vessels across the entireconstruct for both co-culture of mesenchymal progenitors derived fromhiPSC line 1013A and BMSC with HUVEC 4 weeks after seeding. To followthe formation of vascular network in vitro, cell populations werespecifically labeled with different Vybrant® tracker dyes beforeembedding in fibrin clots, and cultured for 4 weeks in a mixture ofosteogenic and endothelial inducing media before harvesting forhistological analysis. No vascular structures were observed when HUVECwere cultured alone, suggesting the pivotal role of mesenchymal cells tosupport and guide tissue vascularization. Studies can be carried out toidentify the molecular mechanism underlying this finding in order todevelop improved protocols to support maturation of vascularized bonetissue in vitro.

Similar outcomes were observed when cells were seeded ontodecellularized bone scaffolds (8 mm Ø×2 mm height) and cultured for 6weeks under osteogenic- and vascular-inducing conditions. The maturationof bone-like tissue, evidenced by the positive staining for osteocalcin,osteopontin and bone sialoprotein, was accompanied by the formation ofnetworks of hollow vessels inside the constructs. Immunohistochemicalexamination showed that the tubular structures were positive for theendothelial marker CD31.

Different seeding ratios, and culture conditions can be tested toexplore the potential to enhance the formation of vascularized bonetissue, as well as to assess the potential of other hiPSC lines forengineering vascularized bone grafts. Future studies are aimed atexploring the effect of dynamic conditions in perfusion bioreactors onthe vascularization process. Development of proper vascularizationprotocols, in combination with the biomimetic osteoinductivescaffold-perfusion bioreactor approach, will allow the construction ofvascularized bone grafts for personalized repair treatments of complexskeletal defects.

Research Design and Methods

This Example proposes the engineering of vascularized bone grafts fromhiPSCs using a stepwise differentiation approach, starting withderivation of lineage-specific osteogenic and endothelial progenitors,and subsequent co-culture of these progenitors in a “biomimetic”scaffold-bioreactor system, which ensure controlled development offunctional bone tissue in vitro. Computer-aided and rapid prototypingtechnologies will be employed to enable the fabrication of custom-madebone substitutes for the reconstruction of large and geometricallycomplex skeletal defects. Engineering patient-specific custom-made bonegrafts can be used to develop innovative treatments to restore skeletalintegrity and functionality in clinical situations characterized bysevere bone loss. This Example describes three sub-projects as describedbelow.

1: Computer-Aided Design (CAD) of Skeletal Models and Computer-AidedManufacturing (CAM) of Biomaterial Scaffolds and Perfusion Bioreactors

The objective of Part 1 is to create and elaborate digital models ofskeletal defects to guide the design and manufacturing of customizedbiomaterial scaffolds and perfusion bioreactors. Digital models ofskeletal defects will be created and segmented into complementarysub-parts using CAD software, then these models will be used as areference for the computer-aided fabrication of biomaterial scaffolds ofcorresponding size and shape and custom-made perfusion bioreactors.Bioreactors will be machined and/or free-form fabricated using thedigital models in order to accommodate each specific cell/scaffoldconstruct in a press-fit fashion and allow culture under directperfusion.

Digital models of skeletal defects will be created using CAD software(e.g., Autocad®, Solidworks®, ProE®, Creo®). To validate the therapeuticpotential of the proposed engineering strategy, this approach can beextended to defect models of different size and shape. Reference modelsof skeletal defects in CAD will be edited and segmented into smallercomplementary sub-parts (lego-like building parts) that can be culturedin perfusion bioreactors without affecting the perfusion system. Thesegmented bone sample files will then be saved in compatible IGES or SLTformats and imported in CAM software (e.g., SprutCAM). The generatedfiles in CAM software will then be processed to generate the appropriateG-Codes to drive a computer-numerical-control (CNC) milling machine(e.g., Tormach, Bridgeport), select appropriate machining tools bits andprogram the machining paths to cut the scaffolding materials into thedesired segmented shapes. Plugs of trabecular bone (cow and/or human) ofadequate size will be drilled, cleansed under high-pressure streamedwater to remove the bone marrow, and then sequentially washed to removecellular material as previously described (41). Decellularized boneplugs will then be freeze-dried, and used for the fabrication ofscaffolds corresponding to the shape and size of the segmented samplesof the skeletal defect. The potential to use synthetic, resorbable andmechanically compliant ceramic/polymer composite materials will beexplored in parallel, since it represents an essential requisite for thereproducible and large-scale fabrication of bone substitutes forclinical applications. Fabricated scaffolds will be sterilized andconditioned in culture medium overnight prior to cell seeding. Thesegmented bone sample files edited in CAD will then be used to designcustomized bioreactor, which can accommodate the cell/scaffoldconstruct(s) in a press-fit fashion under direct perfusion conditions.Again, the CAD files will be converted into compatible formats andimported into CAM and/or 3D printer software, and used to fabricate thebioreactors using different plastic materials. Each bioreactor will beconstituted of two parts (top and bottom) that will be secured together,for example, by means of metallic screws. The cell/scaffold constructswill be cultured in between the top and bottom elements. The bottom partwill include key elements including but not limited to the inlet portand channels for flow perfusion, as well as anatomically shaped chambersto accommodate the cell/scaffold constructs. The top part will includeelements such as a medium reservoir and the outlet port for flowperfusion. A system of tubes can be used to connect the inlet and outletports and allow perfusion throughout the bioreactors via the control ofa peristaltic pump.

2: Engineering Vascularized Bone in Custom-Made Perfusion Bioreactors

The objective of Part 2 is to engineer vascularized patient-specificbone grafts in vitro. hiPSC lines reprogrammed from different tissuesusing non-integrating vectors will be induced toward the mesenchymal andendothelial lineage prior to culture under biomimetic conditions in theosteoinductive scaffold-perfusion bioreactor system as described hereinto guide maturation of functional vascularized bone tissue.

hiPSC reprogrammed using non-integrating vectors from different donorsand source tissues (line BC1 and 1013A) will be expanded, characterizedfor pluripotency and karyotyped before induction toward the mesenchymaland endothelial lineages. Derived progenitor cells will be expanded,characterized by flow cytometry, and karyotyped to assess geneticnormality. Qualitative and quantitative methods will be used to evaluateosteogenic and endothelial phenotype in vitro, including histologicaland immunohistochemical examination, biochemical and morphologicalassays, and gene expression analysis. Vascular induction will be testedin monolayer cultures and embryoid bodies, in the presence of specificfactors (BMP-4, activin, bFGF, VEGF). Differentiated progenitors will besorted based on surface antigen expression (CD34, CD31, KDR, C-KIT) andcultured in endothelial media. Progenitor yield, viability,proliferation and phenotype—expression of specific markers (CD31, vWF,VE-cadherin, SMA) will be assessed by flow cytometry, immunofluorescenceand gene expression. Network formation and sprouting will be tested byencapsulation in collagen/fibronectin/Matrigel before co-cultivationstudies. Commercially available BMSC (Lonza) and HUVEC (Lonza) will beused as reference lines to assess the quality and functionality ofhiPSC-derived mesenchymal and endothelial progenitors. To engineervascularized bone tissue, hiPSC-derived mesenchymal and endothelialprogenitors will be co-seeded onto decellularized bone scaffolds (orothers) and cultured in bioreactor in a mix of osteogenic andendothelial medium. Pre-differentiation, cell seeding ratios,concentration of differentiation factors and use of fibrin sealants willbe explored to design optimal culture conditions for the development offully vascularized bone grafts in vitro. Culture in bioreactors will beconducted for a period of 3-5 weeks, until the formation of a maturevascularized tissue. Tissue development will be assessed usingqualitative and quantitative methods, including histological andimmunohistochemical examination, biochemical assays, high-resolutioncharacterization techniques (SEM, FIB-TEM, Tof-SIMS), imaging procedures(microCT) and mechanical testing (Young's modulus, tensile andcompressive strength).

3: Gluing of Engineered Bone Segments and Evaluation of Stability

The objective of Part 3 is to fabricate custom-made bone grafts forcomplex skeletal reconstruction. Engineered vascularized bone segmentswill be assembled to match the shape of the skeletal defect by means ofa biocompatible bone glue, or reinforced using 3D printed metallic (forexample, titanium) or resorbable pins and holes. Future studies will beaimed at exploring safety and efficacy of engineered bone in animalmodels of critical-sized skeletal defects (both in loading andnon-loading anatomical locations).

Engineered bone segments will be assembled to match the shape of themodel of skeletal defect by means of a biocompatible bone glue forwelding large bone grafts or reinforced using 3D printed metallic (forexample, titanium) or resorbable pins and holes. Future studies will beaimed at exploring the safety and regenerative potential of engineeredbone in animal models of complex critical sized skeletal defects (bothin loading and non-loading skeletal locations). For example, digitalmodels of femoral head defects in adult animals will be created usingmedical imaging procedures (CT scan) and 3D images processed andsegmented (as described above) and used to engineer vascularized bone asdescribed herein. Femoral head ostectomy will then be performed in theanimals to remove the femur head to an extent matching the digital modelgenerated (as described above), and the engineered vascularized boneplaced in site to restore skeletal integrity and functionality. Tissuedevelopment, healing and quality of regenerated tissue will be evaluatedin vivo using medical imaging procedures and following explantationusing histological and immunohistochemical techniques, high-resolutioncharacterization techniques (e.g., SEM, FIB-TEM, Tof-SIMS), andmechanical testing (e.g., Young's modulus, tensile and compressivestrength).

As described herein, vascularized bone grafts can be engineered usingosteogenic and endothelial progenitors derived from human inducedpluripotent stem cells for personalized reconstructive therapies.Although endothelial progenitors can be derived from both hESCs andhiPSCs (38-40), the derivation efficiency is low and the derivedprogenitors display scarce proliferation ability, which limits thepossibility to generate enough cells for engineering large vascularizedbone substitutes. To speed up the development of suitablevascularization protocols, in parallel to optimizing the derivation ofhighly proliferative endothelial progenitors from hiPSCs, commerciallyavailable HUVECs can be used, and then the protocols can be translatedto endothelial progenitors derived from hiPSCs. The hiPSC-derivedmesenchymal progenitors may be expanded to a required amount beforeinduction toward the endothelial lineage, and then used to engineervascularized bone substitutes.

As described herein, the engineered bone substitutes can be assembled tomatch the shape of the skeletal defect using a biocompatible bone gluefor welding large bone grafts, which might be insufficient to ensure astable connection following implantation in high load-bearing locations.To solve this problem, alternative solutions will be tested, includingreinforcement using 3D printed metallic or resorbable pins and holes.

Human Stem Cells

A stepwise protocol is proposed for preparation of vascularized bonegrafts from hiPSCs, which will include: (a) Differentiation andexpansion of osteogenic and vascular progenitors from hiPSCs, andtesting their functional potential for new tissue formation; (b)Preparation and seeding of decellularized bone scaffolds or any otherbiocompatible and resorbable biomaterial scaffolds; and (c) Cultivationof osteogenic tissue phase in conjunction/sequence with formation ofmicrovascular network.

Cell lines: hiPSC lines 1013A (derived by Sendai virus in the NYSCFlaboratory) and BC1 (derived by episomal plasmid vector, from LifeTechnologies) can be used. Initial studies will be done in parallel withESC line H9 (from Wicell Research Institute) and commercially availableadult cells (BMSC and HUVEC from Lonza).

The data generated from this protocol are expected to provide a proof ofconcept for development of vascularized bone substitutes from hiPSCutilizing bioreactors as described herein.

Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. A perfusion bioreactor suitable for use in thepreparation of a tissue graft segment comprising: a) a graft chamber;and b) an equilibration chamber in fluid communication with the graftchamber.
 2. The perfusion bioreactor of claim 1, wherein the graftchamber is configured to accommodate a tissue graft segment.
 3. Theperfusion bioreactor of claim 1, wherein the graft chamber furthercomprises a graft chamber insert configured to accommodate a tissuegraft segment.
 4. The perfusion bioreactor of claim 1, comprising abottom portion and a top portion, the bottom portion comprising: i) thegraft chamber, ii) the equilibration chamber, iii) an inlet, and iv) afluid channel defining a fluid path between the inlet and theequilibration chamber.
 5. The perfusion bioreactor of claim 4, whereinthe top portion comprises: i) a fluid reservoir, ii) an aperture fluidlyconnecting the fluid reservoir and the graft chamber, and iii) an outletport.
 6. The perfusion bioreactor of claim 5, wherein the bottom and topportions are attached via a fastening mechanism.
 7. The perfusionbioreactor of claim 1, further comprising an inlet, a fluid channeldefining a fluid path between the inlet and the equilibration chamber, afluid reservoir, an aperture fluidly connecting the fluid reservoir andthe graft chamber, the fluid reservoir further comprising an outletport.
 8. The perfusion bioreactor of claim 7, wherein the graft chamberis disposed adjacent the equilibration chamber.
 9. The perfusionbioreactor of claim 8, further comprising a fluid pump and one or moretubes fluidly connecting the inlet port and the outlet port to the pumpthereby providing a fluid circuit.
 10. The perfusion bioreactor of claim1, wherein the graft chamber is custom-designed to accommodate thetissue graft segment.
 11. The perfusion bioreactor of claim 3, whereinthe graft chamber insert is custom-designed to accommodate the tissuegraft segment.
 12. The perfusion bioreactor of claim 1, wherein thegraft chamber is custom-designed to accommodate the tissue graft segmentusing a digital three-dimensional model of the tissue graft segment. 13.The perfusion bioreactor of claim 3, wherein the graft chamber insert iscustom-designed to accommodate the tissue graft segment using a digitalthree-dimensional model of the tissue graft segment.
 14. The perfusionbioreactor of claim 1, wherein the tissue graft segment has a maximumthickness of about one centimeter or less.
 15. The perfusion bioreactorof claim 1, wherein the tissue graft segment has a maximum thickness ofabout 0.3 millimeters to 10 millimeters.
 16. The perfusion bioreactor ofclaim 12, wherein the digital three-dimensional model of the tissuegraft segment is generated by medical imaging, computed tomography,computer-assisted design, or any combination thereof.
 17. The perfusionbioreactor of claim 13, wherein the digital three-dimensional model ofthe tissue graft segment is generated by medical imaging, computedtomography, computer-assisted design, or any combination thereof. 18.The perfusion bioreactor of claim 1, wherein the equilibration chambercomprises a flat floor.
 19. The perfusion bioreactor of claim 1, whereinthe equilibration chamber comprises a tapered floor.
 20. The perfusionbioreactor of claim 19, wherein the taper forms an angle of about 10,20, 30, 40, 50, 60 or 70 degrees between the floor and the equilibrationchamber wall.
 21. The perfusion bioreactor of claim 7, wherein theequilibration chamber further comprises one or more diffusion enhancingelements.
 22. The perfusion bioreactor of claim 7, wherein theequilibration chamber further comprises an insert to maintain thedimensions of the equilibration chamber and/or maintain fluid flowthrough the perfusion bioreactor.
 23. The perfusion bioreactor of claim7, wherein the graft chamber further comprises an insert to (i) maintainthe size and dimensions of the graft chamber and equilibrium chamber,and/or (ii) maintain fluid flow through the perfusion bioreactor. 24.The perfusion bioreactor of claim 6, wherein the fastening mechanismcomprises screws, rods, pins, clips, latches or any combination thereof.25. The perfusion bioreactor of claim 6, further comprising an o-ring orother sealing device capable of preventing fluid leakage from betweenthe bottom and top portions.
 26. The perfusion bioreactor of claim 9,wherein the pump is a peristaltic pump.
 27. The perfusion bioreactor ofclaim 6, further comprising a sealing mechanism situated between the topand the bottom portions.
 28. The perfusion bioreactor of claim 4,wherein the bottom portion and top portion are formed as a singleunitary piece.
 29. The perfusion bioreactor of claim 6, wherein the topand bottom portions further comprise one or more holes to facilitate thefastening mechanism.